Car T-cells recognizing cancer-specific IL 13Ra2

ABSTRACT

Provided are specific binding molecules, or fragments thereof, that bind to an epitope of IL13Rα2, a receptor polypeptide preferentially found on the surface of cancer cells rather than healthy cells. Exemplary specific binding molecules are bispecific binding molecules that comprise a fragment of an IL13Rα2 binding molecule and a peptide providing a second function providing a signaling function of the signaling domain of a T cell signaling protein, a peptide modulator of T cell activation, or an enzymatic component of a labeling system. Also provided are polynucleotides encoding such a specific binding molecule (e.g., bispecific binding molecule), vectors, host cells, pharmaceutical compositions and methods of preventing, treating or ameliorating a symptom associated with a cancer disease such as a solid tumor disease (e.g., glioblastoma multiforme).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/545,950 filed Jul. 24, 2017, which is a national phase applicationunder 35 U.S.C. § 371 of International Application No. PCT/US2016/014985filed Jan. 26, 2016, which claims the priority benefit under 35 U.S.C. §119(e) of Provisional U.S. Patent Application No. 62/107,980, filed Jan.26, 2015 and Provisional U.S. Patent Application No. 62/245,771, filedOct. 23, 2015, the disclosures of which are incorporated herein byreference in their entireties.

INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

This application contains, as a separate part of the disclosure, aSequence Listing in computer-readable form which is incorporated byreference in its entirety.

FIELD OF THE DISCLOSURE

The disclosure relates generally to the fields of cancer biology and tomolecular antibody-receptor technology.

BACKGROUND

Cancer is a major threat to human and non-human animal health, leadingto reduced quality of life and, in too many cases, death. The burdenplaced on national, regional and local healthcare organizations to treatand prevent the various forms of cancer is significant in terms of theresources and manpower required. One of the main weapons vertebrates,including humans, have to combat disease is a functioning immune system.A brief consideration of immunotherapies to treat or prevent cancermight lead one to conclude that the effort held out little hope ofsuccess because immune systems guard against foreign, or non-self,materials and cancer cells arise from within, i.e., they are selfmaterials. Continued progress in our understanding of cancer andimmunology is modifying that view, however.

Mutant antigens are powerful targets for tumor destruction, e.g., inmice, and tumor-infiltrating lymphocytes targeting these mutations causedurable tumor regression in patients. Nevertheless, non-mutant antigenshave been presumed by many scientists to be cancer-specific or“relatively cancer-specific” and safe antigens for vaccine approaches.However, adoptively transferred T cells can be orders of magnitude moreeffective and destructive than vaccinations. As a result, targetingMAGE-A3, HER-2 or CEA with T cells has caused death or serious toxicityin clinical trials now halted (8-11). As was shown in 2002, cancer cellswith extremely high or very low expression levels of a target antigendiffer only in the induction of immune responses, but not at theeffector phase (15).

The high affinity interleukin-13 receptor α2 (IL13Rα2) is selectivelyexpressed at a high frequency by glioblastoma multiforme (GBM) as wellas several other tumor types. One approach for targeting thistumor-specific receptor utilizes the cognate ligand, IL-13, conjugatedto cytotoxic molecules. This approach, however, lacks specificitybecause the lower affinity receptor for IL-13, IL13Rα1, is widelyexpressed by normal tissues.

Most human cancers lack specific antigens that are predictably presentand serve as effective targets for eradication by T cells. Every cancercell type harbors a unique set of mutations causing differenttumor-specific antigens. Identifying an effective unique antigen andisolating an appropriate TCR for transduction of autologous T cells foradoptive immunotherapy is still difficult despite the enormoustechnological progress being made. Adoptive immunotherapy usingantibodies or T cells is clinically as well as experimentally the mosteffective immunotherapy, at least when clinically relevant cancers areconsidered (22). The remarkable success of adoptive immunotherapy withchimeric antibody receptors (CARs) and bispecific T cell engagingproteins (BiTEs) is, however, largely restricted to those specific forCD19/CD20-eradicating B cell malignancies and normal B cells inpatients, i.e., hematopoietic cancers. Thus, there is a need to identifyshared, yet tumor-specific, antigens on a wide range of solid tumors,and a concomitant need to develop prophylactics and therapeutics thatcan diagnose, prevent, treat or ameliorate a symptom of these cancers,along with methods for diagnosing, preventing and treating variouscancers.

SUMMARY

Disclosed herein are T cells expressing a chimeric antigen receptor(i.e., CAR) that specifically recognizes and binds to the α2 Interleukin13 Receptor (i.e., IL13Rα2). The IL13Rα2-specific CARs, generallyreferred to herein as 47-CARs, when expressed in T cells effectivelytarget and kill IL13Rα2-positive target cells. Also disclosed isevidence establishing that 47-CARs with a short spacer region, or SSR(i.e., 47-CAR.SSR), exhibit greater capacity to induce IL2-production inan antigen-dependent manner. Further disclosed herein is experimentalevidence that 47-CAR.SSR T cells have potent anti-tumor activity invivo.

The disclosure provides (i) the sequences of heavy (SEQ ID NO:7) andlight (SEQ ID NO:8) chain variable regions of a monoclonal antibody(i.e., the clone 47 antibody) specifically targeting humantumor-associated antigen, IL13Rα2, and (ii) data demonstrating thefunctionality of the protein encoded by the heavy and light chain cDNAsin the format of an scFv antibody or fusion to other functionalmoieties. The sequences of the heavy and light chain constant regionswere also determined and were found to be identical to the correspondingsequences in Genbank Acc. No. DQ381544.1. In particular, the CH1sequence of the clone 47 antibody is set forth in SEQ ID NO:104, CH2 inSEQ ID NO:105 and CH3 in SEQ ID NO:106; the light chain constant regionsequence of the clone 47 antibody is set forth in SEQ ID NO:107; and thehinge region of the clone 47 antibody in SEQ ID NO:108. The heavy andlight chain can be arranged in different formats, such as single-chainantibody, diabodies, bi- and tri-specific antibodies, fusions withtherapeutic proteins and other moieties, human or humanized wholeantibodies as well as human or humanized Fab fragments and otherfunctional derivatives. The single-chain antibody or other arrangementsof the protein encoded by the heavy and light chains, e.g., a bispecificbinding molecule, may be expressed and conjugated to therapeuticcarriers (e.g., viruses, cells, nanomaterials) for specific delivery oftherapeutic to IL13Rα2-overexpressing tumors or for imaging tumorburden.

Proteins expressed by tumor cells but not by normal cells are attractivemolecules for the selective delivery of cytotoxic molecules.Accordingly, interleukin-13 receptor α2 (IL13Rα2), the high affinityreceptor for interleukin-13 (IL-13), is a promising candidate. IL13Rα2is expressed at a high frequency in the aggressive and incurable form ofprimary brain tumor known as glioblastoma multiforme (GBM) (1-3), aswell as by other solid tumors (4). In contrast, normal tissues expresslittle to no IL13Rα2, with the exception of the testes (6). Notably,IL13Rα1, a different receptor with low affinity for IL-13, is expressedubiquitously by many tissues (7-9), making it a poor candidate forselective targeting of tumor-specific immunotherapeutic applications.

Several studies have investigated the therapeutic properties of an IL-13fusion protein conjugated to a recombinant cytotoxin derived fromPseudomonas exotoxin A (IL-13PE) that induces apoptosis inIL13Rα2-expressing glioma cells in vitro, in preclinical animal models,and in patients tested in clinical trials (17-22). Such agents, however,lack a high specificity of interaction with IL13Rα2 because theyalternatively bind to the ubiquitously expressed IL13Rα1. Therefore,developing highly selective antibody fragments that can be combined witheffectors (e.g., T-cells, toxins) for specificity to IL13Rα2-expressingcells is expected to yield therapeutically beneficial results.

The disclosure captures the tumor specificity of IL13Rα2 by providingprotein binding partners specific for IL13Rα2, rather than mimickingIL13 itself, which would result in a molecule exhibiting a capacity tobind to both IL13Rα1 and IL13Rα2. In addition, the disclosure provides apolynucleotide encoding one of these cancer-specific IL13Rα2 bindingpartners, including polynucleotides comprising codon-optimized codingregions for binding partners specific for an epitope of one of theseIL13Rα2 binding partners. Expressly contemplated are fusion proteins orchimeras that comprise an IL13Rα2 binding partner as defined above inoperable linkage to a peptide providing a second function, such as asignaling function of the signaling domain of a T cell signalingprotein, a peptide modulator of T cell activation or an enzymaticcomponent of a labeling system. Exemplary T cell signaling proteinsinclude 4-1BB (CD137), CD3ζ, and fusion proteins, e.g., CD28-CD3ζ and4-1BB-C3ζ. 4-1BB (CD137) and CD28 are co-stimulatory molecules of Tcells; CD3ζ is a signal-transduction component of the T-cell antigenreceptor. In certain embodiments, the IL13Rα2-specific CAR may beexpressed in two fragments that are inactive without the addition of anexogenous substance. By way of non-limiting example, the CAR wouldconsist of two molecules: 1) the first molecule would contain theIL13Rα2-specific scFc, a hinge, a transmembrane domain, a costimulatorydomain, and a heterodimerizer domain (Exto-TM-HD), and 2) the firstmolecule would contain a transmembrane domain, a costimulatory domain, aheterodimerizer domain, a CD3ζ activating domain (Cyto-HD) (Wu et al;Science. 2015 Oct. 16; 350(6258):aab407). Expression of Exto-TM-HD andCyto-HD in cells would result in an inactive IL13Rα2-CAR unless a smallmolecule, for example but not limited to, a rapalog A/C Heterodimerizeris added that links Exto-TM-HD and Cyto-HD, allowing for pharmacologicalcontrol of IL13Rα2-CAR activity. The peptide or protein providing asecond function may provide a modulator of T cell activation, such asIL15, IL15Rα, of an IL15/IL15Rα fusion, or it may encode a label or anenzymatic component of a labeling system useful in monitoring the extentand/or location of binding, in vivo or in vitro. Agent encoding theseprophylactically and therapeutically active biomolecules placed in thecontext of T cells, such as autologous T cells, provide a powerfulplatform for recruiting adoptively transferred T cells to prevent ortreat a variety of cancers in some embodiments of the disclosure. Codonoptimization of the coding regions for binding partners specific forepitopes found on cancer cells provides an efficient approach todelivery of the diagnostic, prophylactic, and/or therapeutic proteinsdisclosed herein.

In one aspect, the disclosure provides an Interleukin 13 Receptor α2(IL13Rα2) binding partner comprising the antibody heavy chain variablefragment (V_(H)) complementarity determining region 1 (CDR1) of SEQ IDNO:1, the V_(H) CDR2 of SEQ ID NO: 2, the V_(H) CDR3 of SEQ ID NO: 3,the light chain (V_(L)) complementarity determining region 1 (CDR1) ofSEQ ID NO: 4, the V_(L) CDR2 of SEQ ID NO: 5, and the V_(L) CDR3 of SEQID NO: 6, wherein the IL13Rα2 binding partner specifically binds to anepitope of IL13Rα2. In some embodiments, the V_(H) sequence is set forthas SEQ ID NO: 7 and in some of the same and some different embodiments,the V_(L) sequence is set forth as SEQ ID NO: 8.

A related aspect of the disclosure provides a bispecific bindingmolecule comprising a fragment of the IL13Rα2 binding partner describedherein that binds to the IL13Rα2 epitope covalently linked to a peptideproviding a second function to form a bispecific binding molecule. Insome embodiments, the second function of the peptide is selected fromthe group consisting of a signaling function of the signaling domain ofa T cell signaling protein, a peptide modulator of T cell activation,and an enzymatic component of a labeling system. In some embodiments,the fragment is a single-chain variable fragment (scFv), which may becontained within a bi-specific T-cell engager (BiTE) or a chimericantigen receptor (CAR). Some embodiments are provided wherein thebispecific binding molecule as described herein is conjugated to atherapeutic carrier.

Another aspect of the disclosure is drawn to a pharmaceuticalcomposition comprising the IL13Rα2 binding partner as described hereinand a pharmaceutically acceptable carrier, adjuvant or diluent.

A related aspect provides a kit comprising the pharmaceuticalcomposition described herein and a protocol for administration of thecomposition. Also related is an aspect providing a polynucleotideencoding the IL13Rα2 binding partner as described herein and a vectorcomprising the polynucleotide as described herein. Yet another aspect isdirected to a host cell comprising the polynucleotide described hereinor the vector described herein.

Yet another aspect of the disclosure provides a method of preventing,treating or ameliorating a symptom of a cancer disease comprisingadministering a therapeutically effective amount of the pharmaceuticalcomposition as described herein. In some embodiments, the cancer is asolid tumor, such as a glioblastoma multiforme (GBM). In someembodiments, the cancer is treated by inhibiting the growth rate of thesolid tumor. In some embodiments, the symptom ameliorated is pain.

More particularly, one aspect of the disclosure is drawn to anIL13Rα2-specific chimeric antigen receptor (CAR) comprising: (A) each ofthe amino acid sequences of: NYLMN (SEQ ID NO: 1); RIDPYDGDIDYNQNFKD(SEQ ID NO: 2); GYGTAYGVDY (SEQ ID NO: 3); RASESVDNYGISFMN (SEQ ID NO:4); AASRQGSG (SEQ ID NO: 5); and QQSKEVPWT (SEQ ID NO: 6), (B) a hingeregion, (C) a transmembrane domain, and (D) an endodomain comprising asignaling domain a CD3 zeta chain and a signaling domain of CD28. Insome embodiments, the endodomain further comprises a signaling domain ofone or more of: CD137, CD134, CD27, CD40, ICOS, and Myd88, optionally,wherein the endodomain comprises one or more of the amino acid sequencesof SEQ ID NOs: 68, 70, 72, 74, 76, and 78. In some embodiments, thehinge region comprises the amino acid sequence of SEQ ID NO: 35 or SEQID NO: 37. In some embodiments, the IL13Rα2-specific CAR comprises atransmembrane domain of CD28. In some embodiments, the IL13Rα2-specificCAR comprises the amino acid sequence of SEQ ID NO: 39. In someembodiments, the CD3 zeta chain signaling domain comprises the aminoacid sequence of SEQ ID NO: 41. In some embodiments, theIL13Rα2-specific CAR comprises the amino acid sequence of SEQ ID NO: 47.In some embodiments, the IL13Rα2-specific CAR comprises the amino acidsequence of SEQ ID NO 49 or 51. In some embodiments, theIL13Rα2-specific CAR comprises one or both of the amino acid sequencesof SEQ ID NO: 7 and/or SEQ ID NO: 8. In some embodiments, theIL13Rα2-specific CAR of claim 9, wherein the amino acid sequence of SEQID NO: 7 is fused to the amino acid sequence of SEQ ID NO: 8 through alinker. In some embodiments, the linker comprises the amino acidsequence of EEGEFSEAR (SEQ ID NO 10). In some embodiments, theIL13Rα2-specific CAR comprises the amino acid sequence of SEQ ID NO: 13.In some embodiments, the IL13Rα2-specific CAR comprises the amino acidsequence of SEQ ID NO: 53 or SEQ ID NO: 55.

In a related aspect, the disclosure provides a nucleic acid encoding anyof the IL13Rα2-specific CARs disclosed or described herein. In someembodiments, the nucleic acid comprises the sequence of SEQ ID NO: 34,36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, or 65.

In yet another aspect, the disclosure provides a vector comprising anucleic acid disclosed or described herein. In some embodiments, thevector is a retroviral vector.

In another aspect, the disclosure provides a host cell comprising avector disclosed or described herein. In some embodiments, the host cellis a human host cell. In some embodiments, the host cell is aT-lymphocyte. In some embodiments, the host cell is a natural killercell.

In a related aspect, the disclosure provides a cell populationcomprising a host cell disclosed or described herein. In someembodiments, the cell population comprises at least 10⁷ host cells.

Another aspect is drawn to a pharmaceutical composition comprising anIL13Rα2-specific CAR as disclosed or described herein, a nucleic acid asdisclosed or described herein, a vector as disclosed or describedherein, a host cell as disclosed or described herein, or a cellpopulation as disclosed or described herein, and a pharmaceuticallyacceptable carrier.

Another aspect of the disclosure provides a method of treating a cancerin a subject, comprising administering to the subject a cell populationas disclosed or described herein, in an amount effective to treat thecancer in the subject. In some embodiments, the cancer is colon cancer.In some embodiments, the host cells of the cell population are cellsobtained from the subject. In some embodiments, the cells obtained fromthe subject are T-lymphocytes. In some embodiments, the cells obtainedfrom the subject are natural killer cells.

Another aspect of the disclosure provides an IL13Rα2-specific chimericantigen receptor (CAR) comprising: (A) an ectodomain comprising each ofthe amino acid sequences of: (i) NYLMN (SEQ ID NO: 1); (ii)RIDPYDGDIDYNQNFKD (SEQ ID NO: 2); (III) GYGTAYGVDY (SEQ ID NO: 3); (iv)RASESVDNYGISFMN (SEQ ID NO: 4); (v) AASRQGSG (SEQ ID NO: 5); and (vi)QQSKEVPWT (SEQ ID NO: 6); (B) a spacer region; (C) a transmembranedomain; and (D) an endodomain selected from the group consisting ofCD3.ζ, CD28.ζ, CD28.OX40.ζ, CD28.41BB.ζ and 41BB.ζ. In some embodiments,the spacer region comprises no more than 100 amino acids, or no morethan 50 amino acids, or no more than 25 amino acids, or the spacerregion comprises SEQ ID NO:103 (PKSCDKTHTCPPCPAPEL) from the IgG1 hingeregion. In some embodiments, the transmembrane domain comprises thetransmembrane domain of CD28, such as a transmembrane domain comprisingthe amino acid sequence of SEQ ID NO:39, or CD8α. In some embodiments,the endodomain further comprises a signaling domain of one or more of:CD137, CD134, CD27, CD40, ICOS, and Myd88. In some embodiments, theendodomain comprises one or more of the amino acid sequences of SEQ IDNOs: 68, 70, 72, 74, 76, and 78. In some embodiments comprising the CD3zeta chain signaling domain, the IL13Rα2-specific CAR comprises theamino acid sequence of SEQ ID NO: 41. In some embodiments, theIL13Rα2-specific CAR comprises the amino acid sequence of SEQ ID NO: 47.In some embodiments, the IL13Rα2-specific CAR comprises the amino acidsequence of SEQ ID NO 49 or 51. In some embodiments, theIL13Rα2-specific CAR comprises one or both of the amino acid sequencesof SEQ ID NO: 7 and/or SEQ ID NO: 8.

The disclosure also contemplates embodiments wherein the amino acidsequence of SEQ ID NO: 7 is fused to the amino acid sequence of SEQ IDNO: 8 through a linker. In some embodiments, the linker comprises theamino acid sequence of EEGEFSEAR (SEQ ID NO 10). In some of theseembodiments, the IL13Rα2-specific CAR comprises the amino acid sequenceof SEQ ID NO: 13. In some embodiments, the IL13Rα2-specific CARcomprises the amino acid sequence of SEQ ID NO: 53 or SEQ ID NO: 55.

Another aspect of the disclosure is drawn to a nucleic acid encoding theIL13Rα2-specific CAR disclosed herein. In some embodiments, the nucleicacid comprises the sequence of SEQ ID NO: 34, 36, 38, 40, 42, 44, 46,48, 50, 52, 54, 56, and 65.

Still another aspect of the disclosure is drawn to a vector comprisingthe nucleic acid disclosed herein. In some embodiments, the vector is aretroviral vector.

Yet another aspect of the disclosure is a host cell comprising thevector disclosed herein. In some embodiments, the host cell is a humanhost cell. In some embodiments, the host cell is a T-lymphocyte or anatural killer cell. In some embodiments, the cells obtained from thesubject are T cells, and/or other lymphocytes including, but not limitedto, NKT cells, γδ T cells, mucosa associated invariant T cells or MAITcells, and innate lymphoid cells. In addition, stem and/or progenitorcells may be obtained from the subject that are subsequentlydifferentiated into the aforementioned immune cells.

Another aspect of the disclosure is a cell population comprising thehost cell disclosed herein. In some embodiments, the cell populationcomprises at least 10⁷ host cells.

In another aspect, the disclosure provides a pharmaceutical compositioncomprising an IL13Rα2-specific CAR as disclosed herein, a nucleic acidas disclosed herein, a vector as disclosed herein, a host cell asdisclosed herein, or a cell population as disclosed herein, and apharmaceutically acceptable carrier.

Yet another aspect of the disclosure is a method of treating a cancer ina subject, comprising administering to the subject a cell population asdisclosed herein, in an amount effective to treat the cancer in thesubject. In some embodiments, the cancer is colon cancer. In someembodiments, the host cells of the cell population are cells obtainedfrom the subject. In some embodiments, the cells obtained from thesubject are T cells, and/or other lymphocytes including, but not limitedto, NKT cells, γδ T cells, mucosa associated invariant T cells or MAITcells, and innate lymphoid cells. In addition, stem and/or progenitorcells may be obtained from the subject that are subsequentlydifferentiated into the aforementioned immune cells.

In some embodiments, the immune or stem and/or progenitor cells that aregenetically modified to be IL13Rα2-specific by expressing a CAR or BITEmolecule may be further genetically modified to enhance their anti-tumoractivity. Non-limiting examples of additional genetic modificationinclude, but are not limited to: i) CARs or BITEs that are specific forother antigens expressed on tumor cells or within the tumor environment,ii) cytokines (e.g., various interleukins such as IL7, IL12, IL15,IL21), iii) chimeric cytokine receptors (e.g., IL7R, IL15R), iv)chemokine receptors (e.g., CCR2b, CXCR2), iv) chimeric activatingreceptors (e.g., IL4/IL7R, IL4/IL2R, TGFβ/TLR4R), v) silencing negativeregulators (e.g., PD1, SHP1), vi) silencing endogenous TCR expression,and vii) inducible suicide genes (e.g., CD20, truncated EGFR, induciblecaspase 9).

Other features and advantages of the disclosure will become apparentfrom the following detailed description, including the drawing. Itshould be understood, however, that the detailed description and thespecific examples, while indicating preferred embodiments, are providedfor illustration only, because various changes and modifications withinthe spirit and scope of the invention will become apparent to thoseskilled in the art from the detailed description.

BRIEF DESCRIPTION OF THE DRAWING

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1A-D. Characterization of antigen recognition and screening ofhybridoma clones. A, binding of B-D13 mAb to ELISA plates coated withrhIL13Rα2hFc at 0.1 and 1 μg/ml. B, binding of IL13Rα2 mAb to native anddenatured (at 95° C. in the presence of β-mercaptoethanol) rhIL13Rα2hFcin a plate-bound ELISA. A paired t test was used to evaluate thedifference between control groups (n=4). *, p<0.1; ***, p<0.001. Errorbars represent S.D. These data are representative of two independentexperiments. C, screening of selected hybridoma populations againstrhIL13Rα2hFc in a plate-bound ELISA. D, screening of selected hybridomapopulations against rhIL13Rα2hFC using a Western blot.

FIG. 2A-D. The IL13Rα2 (clone 47) mAb specifically binds to rhIL13Rα2and IL13Rα2 expressed on the cell surface of CHO cells. A, binding ofIL13Rα2 (clone 47, 83807, and B-D13) mAbs to rhIL13Rα2 in a plate-boundELISA. B, binding of the IL13Rα2 (clone 47) mAb to human IL13Rα2expressed on the surface of CHO cells. C, cross-reactivity of theIL13Rα2 (clone 47) mAb with hrIL13Rα1. D, cross-reactivity of IL13Rα2(clones 47, 83807, and B-D13) mAbs with mouse rIL13Rα2. Error barsrepresent S.D.

FIG. 3A-D. Binding of IL13Rα2 mAb to glioma cells. A, flow charts ofIL13Rα2 (clones 47, 83807, and B-D13) mAbs binding to the surface ofglioma cells, normal human primary astrocytes, and HEK cells transfectedwith IL13Rα2. B, data of the median fluorescence intensity of bindingbetween the IL13Rα2 (clones 47, 83807, and B-D13) mAbs to various celllines analyzed by flow cytometry. Numbers above the bars represent thedifference in the binding of clone 47 when compared with clone B-D13 foreach cell line. The color key is the same for A and B. C, mRNAexpression for IL13Rα2 in glioma cells as well as normal human primaryastrocytes. D, panels a-c, flow cytometry demonstrating the specificbinding of the IL13Rα2 (clone 47) mAb to GFP-tagged U251 glioma cellsfrom an intracranial xenograft (xeno). The curve with a clear area underthe curve in sub-panel b depicts the binding of mAb IL13Rα2 (clone 47)to GFP negative cells; the curve with a clear area under the curve insub-panel c depicts the binding of mAb IL13Rα2 (clone 47) to GFPpositive cells. Curves in sub-panels b and c with gray areas under thecurves show the results when exposing control IgG to GFP-negative(sub-panel b) or GFP-positive (sub-panel c) cells. neg, negative. A,area; SSC-A, side scatter area; APC-A, allophycocyanin area.

FIG. 4A-C. The affinity between the IL13Rα2 (clone 47) mAb andrhIL13Rα2. The kinetics of interaction of IL13Rα2 (clone 47) mAb (A) andthe commercially available mAb clones 83807 (B) and B-D13 (C) withrhIL13Rα2 as visualized by SPR in a Biacore 3000 are shown. TherhIL13Rα2 was injected at concentrations ranging from 1 to 100 nM (1 nM,2.5 nM, 5 nM, 7.5 nM, 10 nM, 15 nM, 20 nM, 25 nM concentrations shown,lower to upper curves) at a constant flow rate of 20 μl/minute overimmobilized antibodies and over a control dextran surface (these valueswere subtracted from the signal). The association and dissociationphases were monitored for 300 s by following the change in SPR signal(colored curves) given in RU. Black curves represent the fit of the datato a one-site binding model. For derived kinetic parameters, seeTable 1. Lower panels show residuals from a one-site binding model,indicating an excellent fit.

FIG. 5A-C. The IL13Rα2 (clone 47) mAb competes with rhIL-13 for thebinding site of IL13Rα2. A, using a competitive binding plate assay, theIL13Rα2 (clone 47) mAb but not control mIgG or antibody clones 83807,B-D13, and YY-23Z significantly abolished the binding of rhIL-13 to therhIL13Rα2Fc chimera absorbed to plastic. One-way analysis of variancefollowed by Dunnett's post hoc test was performed. Data from a singlerepresentative experiment are shown. B, recombinant human IL-13 competeswith the IL13Rα2 (clone 47) mAb for the binding site of WT IL13Rα2 butnot with the 4-amino acid (4aa) mutant IL13Rα2 expressed on the surfaceof HEK cells. C, the IL13Rα2 (clone 47) mAb competes with rhIL-13 forthe binding site of the WT and 4-amino acid mutant form of IL13Rα2. Apaired t test was performed. Data represent the summary of threeindependent experiments shown in B and C. *, p<0.05; **, p<0.01; ***,p<0.001. Error bars represent S.D.

FIG. 6A-B. The contribution of Tyr207, Asp271, Tyr315, and Asp318residues of IL13Rα2 to the binding of the IL13Rα2 (clone 47) mAb. A,variants of cDNA encoding individual mutations to Ala or a combinatorial4-amino acid mutant (4aa mut) of IL13Rα2 was generated. HEK cells weretransfected with a control vector or a vector encoding the IL13Rα2variants. After 48 hours, binding of the IL13Rα2 (clone 47) mAb to thesurface of transfected cells was analyzed by flow cytometry.Anti-IL13Rα2 antibody clones 83807 and B-D13 were used as referenceantibodies in this assay. Binding of antibodies was determined as thepercentage of positive cells. The ratio of bound clones was determinedfor each IL13Rα2 mutant and compared with that of the wild-typereceptor. One-way analysis of variance followed by Dunnett's post hoctest was performed. Data represent a summary of four independentexperiments. Error bars represent S.D. B, representative graphs of flowcytometry data demonstrating the binding of clone 47 or rhIL-13 to theWT and 4-amino acid-mutated variant of the IL13Rα2 receptor expressed onthe surface of HEK cells. Filled curves: negative control, staining withisotype control IgG+secondary antibody; Open curves: staining with theanti-IL13Rα2 (clone 47) monoclonal antibody+secondary antibody. A, area;APC-A, allophycocyanin area; FITC-A, fluorescein isothiocyanate area.

FIG. 7A-C. Effect of N-linked glycosylation on the binding of IL13Rα2 torecombinant IL13Rα2. A, binding of IL13Rα2 to control and PngaseF-treated rhIL13Rα2. Plates were coated with hrIL13Rα2 at 1 μg/ml andtreated with native buffer or with 1 milliunit/well Pngase F in nativebuffer for 3 hours at 37° C. An ELISA for binding of the IL13Rα2 (clone47) mAb in comparison with antibody clones B-D13, 83807, and YY-23Z andrhIL-13 was performed, and the data of one representative experimentfrom three independent experiments are shown. A paired t test was usedto evaluate the difference between control and Pngase F-treated groups(n=4). *, p<0.5; **, p<0.01; ***, p<0.001. B, a Western blot shows thelower molecular weight of Pngase F-treated rhIL13Rα2 due to removal ofN-linked glycosylation adducts from the molecule. C, flow cytometryshows the binding of IL13Rα2 mAbs to IL13Rα2-expressing U251 and HEK293cells treated with 1 milliunit of Pngase F for 1 hour at 37° C. The dataare representative of three independent experiments. A paired t test wasused to evaluate the difference between control and Pngase F-treatedgroups. *, p<0.5. MFI, mean fluorescence intensity. Error bars representS.D.

FIG. 8 . The IL13Rα2 (clone 47) mAb recognizes IL13Rα2 in GBM tissuesand in a human glioma xenograft. Immunohistochemistry on frozen tissuesections from three human GBM samples and a U251 xenograft was performedwith the IL13Rα2 (clone 47) mAb or mIgG at a concentration of 3 μg/ml.Staining of GBM tissues demonstrates positive staining of the majorityof cells in sample 1, positive reactivity in only a fraction of thecells in sample 2, and negative staining in sample 3. Staining in allthree samples was performed in the same experiment. Positive stainingwas also detected in U251 xenograft tissue. Arrows point to individualpositive cells. Scale bars=100 μm.

FIG. 9A-B. The IL13Rα2 (clone 47) mAb improves the survival of mice inan orthotopic human glioma xenograft model. A, the survival of animalsinjected with U251 glioma cells (2.5×10⁴) alone or in combination witheither control IgG or the IL13Rα2 (clone 47) mAb. B, a representativephotomicrograph of 10-μm-thick tissue sections stained with H&E frommice injected with U251 cells alone (panels a and b) or in combinationwith mIgG (panels c and d) or mAbIL13Rα2 (clone 47) (panels e and f).Arrows point to the tumor and invading cells. Scale bars (panels a, c,and e)=100 μm. Scale bars (panels b, d, and f)=100 μm.

FIG. 10A-B. A competitive binding assay for the IL13Rα2 (clone 47) mAbto the surface of N10 glioma cells. A. The IL13Rα2 (clone 47) mAb waspre-incubated with 10× excess rhIL13Rα2 for 30 minutes on ice. N10 cellswere subsequently incubated with isotype control mIgG or IL13Rα2 (clone47) mAb alone or in the presence of rhIL13Rα2 and bound antibodies wereanalyzed by flow cytometry. B. N10 glioma cells were pre-incubatedeither with 10× excess rhIL13 (left panel) or with 10× excess of IL13Rα2(clone 47) mAb for 30 minutes on ice (right panel). N10 cells weresubsequently incubated with isotype control mIgG, IL13Rα2 (clone 47) mAbor rhIL13. Bound antibodies or rhIL13 were detected with secondaryantibodies and analyzed by flow cytometry. Data are presented as % ofpositive cells.

FIG. 11 . The effects of IL13Rα2 (clone 47) mAb on the survival of micewith an established human U251 glioma. Mice were intracranially-injectedwith 2.5×10⁴ U251 glioma cells and treated three days later with asingle injection of PBS (n=7) or 10 μg IL13Rα2 (clone 47 or B-D13) mAb(n=7). The analysis of the animal's survival was performed using theLog-rank test. Median survival was determined to be 27 days in the PBSgroup, versus 23 and 35 days in the groups treated with B-D13 and 47IL13Rα2 mAb, respectively (p>0.05).

FIG. 12 . Binding of IL13Rα2 clone 47 phages with IL13Rα2hFc in plateELISA. These data demonstrate that phages presenting scFv IL13Rα2 (clone47) are positively selected against IL13Rα2Fc chimeric protein after 3rounds of biopanning.

FIG. 13 . Specificity of binding scFv IL13Rα2 clone 47 withIL13Rα2hFc-competitive assay. These data show that binding of thescFvIL13Rα2 (clone 47) presented on the phage surface to recombinantIL13Rα2 is completely abolished by parental monoclonal antibody (clone47), but not other antibodies against IL13Rα2. It indicates thatscFvIL13Rα2 (clone 47) and parental monoclonal antibody (clone 47) sharethe epitope (i.e., recognition site) on the IL13Rα2 molecule. Each datapoint is an average of 3 independent replicates in all figures. Datapresented as mean±SEM. ***p<0.001.

FIG. 14 . Binding of soluble scFv IL13Rα2 (clone 47) with IL13Rα2hFcchimera. These data show that soluble scFvIL13Rα2 (clone47) generated ina prokaryotic expression system (E. coli) binds specifically toIL13Rα2Fc recombinant protein. Parental antibody, mAb IL13Rα2 (clone47), and control mouse IgG served as positive and negative controls,respectively

FIG. 15 . The effect of mesenchymal stem cells secretingscFvIL13Rα2-sTRAIL fusion protein on the U87-IL13Rα2 glioma cell line.These data show that mesenchymal stem cells modified to secrete agenetic fusion of scFvIL13Rα2(clone 47) with TRAIL protein exhibit atherapeutic effect in the IL13Rα2-expressing U87 glioma cell line. Theresults establish the efficacy of conjugating the scFV to a TRAILcytokine. The amount of cancer cell killing is equivalent to the use ofTRAIL alone without the scFV, but it is expected that the scFV-TRAILwould be less harmful to non-cancer tissues, given the specificityconferred by the scFv targeting IL13Rα2.

FIG. 16 . Schematic maps of retroviral vector encoding IL13Rα2-specificscFv CARs. The CAR consists of the immunoglobulin heavy-chain leaderpeptide, the IL13Rα2-specific scFv clone 47 (M47), a short hinge (SH) orlong hinge (LH), a transmembrane domain (TM) derived from CD28, and aCD28. ζ endodomain. LTR: long terminal repeat (retroviral backbone).Domains are identified as block structures. Maps are not to scale.

FIG. 17 . IL13Rα2-scFv CART cell agent: Expression of αCD3.ζ relative toαGAPDH of CAR agent in T cells. SH: short hinge. LH: long hinge.

FIG. 18A-B. IL13Rα2-scFv CARs are expressed on the surface of T cells.IL13Rα2-CAR T cells were generated by retroviral transduction and CARexpression was determined by FACS analysis. Short hinge CARs weredetected with an antibody specific for murine scFV. Long hinge CARs weredetected with an antibody specific for the long hinge. Isotype antibodycontrol: open curve; Specific Antibody: filled curve.

FIG. 19 . Functional characterization of IL13Rα2-CAR Tcells—Cytotoxicity. Standard ⁵¹Chromium cytotoxicity assays wereperformed with Raji (IL13Rα1−/IL13Rα2−), 293T (IL13Rα1+/IL13Rα2−), 293Tgenetically modified to express IL13Rα2cells (293T-IL13Rα2;IL13Rα1+/IL13Rα2+), or U373 (IL13Rα1+/IL13Rα2+) cells as targets. Aseffectors nontransduced (NT) T cells, IL13Rα2-CAR.SH.CD28.ζ T cells,IL13Rα2−CAR.LH.CD28ζ T cells, IL13Rα2-CAR.SH.Δ T cells, orIL13Rα2-CAR.LH.Δ T cells were used. Only IL13Rα2-CAR.SH.CD28.ζ T cellsand IL13Rα2-CAR.LH.CD28.ζ T cells killed with IL13Rα2+ target cells(U373 and 293T-IL13Rα2; n=4). T cells expressing nonfunctional CARs(IL13Rα2-CAR.SH.4 and IL13Rα2-CAR.LH.Δ) had not cytolytic activity,demonstrating that the killing activity depends on the expression of afunctional IL13Rα2-CAR. NT T cells killed none of the targets, furtherconfirming specificity.

FIG. 20 . Functional characterization of IL13Rα2-CAR T cells—IFNγ andIL2 Cytokine secretions. A. NT T cells, IL13Rα2-CAR.SH.CD28ζ T cells,IL13Rα2-CAR.LH.CD28.ζ T cells, IL13Rα2-CAR.SH.Δ T cells, orIL13Rα2-CAR.LH.Δ T cells were co-cultured with U373 cells for 24 to 48hours (n=4). Only IL13Rα2-CAR.SH.CD28.ζ T cells andIL13Rα2-CAR.LH.CD28.ζ T cells secreted IFNγ demonstrating target cellrecognition in contrast to IL13Rα2-CAR.SH.Δ T cells, IL13Rα2-CAR.LH.Δ Tcells or NT T cells. B. NT T cells, IL13Rα2-CAR.SH.CD28.ζ T cells,IL13Rα2-CAR.LH.CD28.ζ T cells, IL13Rα2-CAR.SH.Δ T cells, orIL13Rα2-CAR.LH.Δ T cells were co-cultured with U373 cells for 24 to 48hours (n=4). Only IL13Rα2-CAR.SH.CD28.ζ T cells secreted IL2,demonstrating that IL13Rα2-CAR.SH.CD28.ζ induces superior T cellactivation in comparison to IL13Rα2-CAR.LH.CD28.ζ. IL13Rα2-CAR.SH.Δ Tcells, IL13Rα2-CAR.LH.Δ T cells or NT T cells also did not induce IL2production.

FIG. 21 . IL13Rα2-SH CARs have anti-glioma activity in vivo. Severecombined immunodeficient (SCID) mice were injected with 1×10⁵ fireflyluciferase expressing U373 cells intracranially. On day 7 mice weretreated either with 1×10⁶ IL13Rα2-CAR.SH.CD28.ζ T cells,IL13Rα2-CAR.LH.CD28.ζ T cells, IL13Rα2-CAR.SH.Δ T cells, orIL13Rα2-CAR.LH.Δ T cells (5 mice per group). Tumor growth was monitoredby bioluminescence imaging. Only IL13Rα2-CAR.SH.CD28.ζ T cells hadsignificant anti-glioma effects with 4/5 mice having a completeresponse.

FIG. 22 . Properties of m47 CAR T cell agent. The m47-CAR T cellsrecognize IL13Rα2⁺, but not IL13Rα1⁺ targets. The data show that theshort hinge CD28z-CAR (SH2) T cells perform better in terms of effectorfunction than CD28z-CAR (SH3), CD28z-CAR (LH2), CD28z-CAR (LH3),CD28z-CAR (SH2Δ), or CD28z-CAR (SH3Δ).

FIG. 23 . Functional comparison of m47 CAR T cell agents. Open curve:secondary antibody; Filled curve: IL13Rα2Fc+ secondary antibody.

FIG. 24 . The m47 CAR T cell agent is highly expressed aftertransduction. Open curve: secondary antibody; Filled curve: IL13Rα2Fc+secondary antibody.

FIG. 25 . The m47 CAR T cell produce interferon γ and interleukin 2, butonly after IL13Rα2 stimulation.

FIG. 26 . IL13Rα2− and IL13Rα1-positive cell lines are made by geneticmodification of HEK 293T cells. Filled curve: isotype antibody control;Open curve: specific antibody.

FIG. 27 . The m47 CAR T cells kill only IL13Rα2⁺ cell lines. The invitro experiments provide data establishing that m47 CAR T cells presenta recombinant CAR protein on the cell surface that does not recognizeIL4R, IL13Rα1 or any receptor other than its specific recognition ofIL13Rα2. The specificity of the recognition extends to a specificity foronly those cell lines expressing IL13Rα2.

FIG. 28 . In vivo data comparing effect of m47 CAR T cell agent,untreated and NT-treated glioblastoma multiforme xenografts in nudemice. The U373 glioblastoma multiforme xenograft mouse model was used.At day 0, 1×10⁵ GFP-ffluc U373 cells were administered per mouse. On day7, 2×10⁶ m47 CAR T cells or NT cells were administered. Untreatedsamples did not receive treatment on day 7. No exogenous interleukin 2was administered and results of the survival analysis were recorded byserial bioluminescence imaging. n=3.

FIG. 29 . The m47 CAR T cell agent prolonged the survival of nude micewith glioblastoma multiforme.

FIG. 30A-C. Characterization of IL13Rα2-CAR T cells. (A, B) Co-cultureassay with recombinant protein demonstrated interferon γ and interleukin2 production in an IL14Rα2-dependent fashion; (C) Cytolytic activity instandard chromium release assay.

FIG. 31A-D. Generation of 47 CAR T cells. (A) Scheme of M47 CARs. AllCARs contained an N-terminal leader sequence, a codon-optimizedsynthetic gene encoding M47 in scFv format, a spacer region, a CD28transmembrane domain, and signaling domains derived from CD28 and CD3-ζ.The spacer region was either the IgG1 hinge (16 amino acids; shortspacer region; M47-CAR.SSR.CD28.ζ) or the IgG1-CH2CH3 domain. LSR.Δ andSSR.Δ M47-CARs without signaling domains were constructed and served ascontrols. (C,B) CAR expression was confirmed using FACS analysis.Representative plots (B) and summary data (C) are shown (mean74.1%-93.3%, n=5-6 per CAR construct). Open curve: secondary antibody;Filled curve: IL13Rα2Fc+ secondary antibody. (D) Expression offull-length 47-CAR.SSR.CD28.ζ and 47-CAR.LSR.CD28.ζ by Western blotanalysis using a CD3-ζ antibody.

FIG. 32 . Phenotypic analysis of 47-CAR T cell lines. CAR T cells wereanalyzed for CD4 and CD8 surface expression using CD4-PacBlue andCD8-PerCP antibodies (BD Biosciences). The four CAR T cell linesanalyzed for surface expression of CD4 and CD8 were SSR.Δ, SSR.CD28.ζ,LSR.Δ, and LSR.CD28.ζ. The histogram provides the results of theanalysis, with light gray bars indicating CD4 expression and black barsindicating CD8 expression.

FIG. 33A-D. 47-CAR T cells release cytokines after stimulation withrecombinant IL13Rα2 protein or IL13Rα2-positive cells. 47-CAR ornon-transduced (NT) T cells were stimulated with recombinant IL13Rα1,IL13Rα2, or IL4Rα proteins. After 24 hours, IFNγ (A) was measured byELISA (n=4). T cells expressing 47-CAR constructs, but not controls,expressed significant levels of IFNγ (p<0.001) when stimulated withrecombinant IL13Rα2 protein in comparison to IL13Rα1 and IL4Rαstimulated T cells. 47-CAR T cells were co-cultured with Raji, U373cells, 293T-GFP, and 293T-GFP/IL13Rα2 at a 1:2 E:T ratio. NT and CAR.Δ Tcells served as controls. (B,C) After 24 hours, cytokines (IFNγ, IL2)were measured by ELISA (n=3). (B) U373 and 293T-GFP-IL13Rα2 (IFNγ);SSR.Δ vs SSR.CD28. ζ: p<0.001; LSR.Δ vs LSR.CD28.ζ: p<0.05. (C); U373and 293T-GFP-IL13Rα2 (IL2); SSR.Δ vs SSR.CD28. ζ: p<0.01; LSR.Δ vsLSR.CD28. ζ: NS. (D) 4-hour cytotoxicity assay at an E:T ratio of 10:1(n=4).

FIG. 34 . LSR.CD28.ζ T cells show a self-activation phenotype during exvivo expansion. T cells were analyzed for phosphor-CD3-ζ expressionusing CD247 (pY142)-AF647 antibody (BD Biosciences).

FIG. 35 . Cell surface expression of IL13Rα1 and IL13Rα2. Cell lineswere analyzed for IL13Rα1 and IL13Rα expression using primary goatanti-IL13Rα1 and anti-IL13Rα2 antibodies (AF152 and AF146, respectively;R&D) followed by secondary rabbit anti-goat IgG Alexa647 antibody (LifeTechnologies). Filled curve: isotype antibody control; Open curve:specific antibody.

FIG. 36A-D. Generation of SSR 47-CARs with CD28.OX40.ζ, CD28.41BB.ζ or41BB.ζ endodomains. (A) Scheme of SSR 47-CARs. (B, C) CAR expression wasconfirmed using FACS analysis. Representative plots (B) and summary data(C) are shown. 47-CAR.SSR.CD28.OX40.ζ and 47-CAR.SSR.CD28.41BB.ζ: mean:74.6%-77.5% (n=4); 47-CAR.SSR.CD28.41BB.ζ: mean: 4.9% (n=3). Open curve:secondary antibody; Filled curve: IL13Rα2Fc+ secondary antibody. (D)Expression of 47-CAR.SSR.41BB.ζ, M47-CAR.SSR.OX40.CD28.ζ andM47-CAR.SSR.41BB.CD28.ζ by Western blot analysis.

FIG. 37A-B. Comparison of 47-CAR.SSR.CD28.ζ 47-CAR.SSR.41BB.ζ and47-CAR.SSR.CD28.OX40.ζ T cells. (A) 47-CAR T cells were co-cultured withU373 cells at a 1:2 E:T ratio. NT and CAR.Δ T cells served as controls.After 24 hours, IFNγ and IL2 were measured by ELISA (n=3); SSR.Δ vsSSR.CD28.ζ (U373; IFNγ): p<0.001; SSR.Δ vs SSR.41BB.ζ (U373; IFNγ):p<0.05; SSR.Δ vs SSR.CD28.OX40.ζ for (U373; IFNγ): p<0.001; SSR.Δ vsSSR.CD28.ζ (U373; IL2): p<0.001; SSR.Δ vs SSR.41BB.ζ (U373; IL2):p<0.001; SSR.Δ vs SSR.CD28.OX40.ζ (U373; IL2): p<0.01. (B) 4-hourcytotoxicity assay at an E:T ratio of 10:1 (n=4).

FIG. 38A-C. Treatment of glioma xenograft with T cells expressing47-CARs results in tumor regression and improved overall survival. U373glioma bearing mice were treated on day 7 with SSR.CD28.ζ (n=9),SSR.41BB.ζ (n=9) or SSR.OX40.CD28.ζ (n=9) T cells. SSR.Δ CAR T cells(n=7) served as controls. (A) Representative images for each group and(B) quantitative bioluminescence (radiance=photons/sec/cm²/sr) imagingdata for all mice are shown (dotted lines: individual mice; solid lines:median). (C) Kaplan-Meier survival analysis (SSR.Δ vs SSR.CD28. ζ:p=0.0002; SSR.Δ vs SSR.41BB.ζ: p=0.0039; SSR.Δ vs SSR.OX40.CD28. ζ:p=0.0092; SSR.CD28. vs SSR.41BB.ζ: p=0.4723; SSR.CD28.ζ vsSSR.OX40.CD28.ζ: p=0.3582; SSR.41BB.ζ vs SSR.OX40.CD28.ζ: p=0.8374).

FIG. 39A-B. Analysis of U373 cells isolated from recurrent tumors. U373cells were isolated from recurrent tumors of mice that were treated with47-CAR T cells. After short-term culture (2 to 7 days), FACS analysisand cytotoxicity assays were performed. (A) FACS analysis for IL13Rα2.(B) 47-CAR T cells killed U373 tumor cells isolated from recurrenttumors in contrast to Raji cells in a standard four-hour cytotoxicityassay for Cr release from labeled cells. Open curve: isotype antibodycontrol; Filled curve: specific antibody.

FIG. 40A-C. Limited persistence of 47-CART cell in vivo.47.SSR.CD28.ζ-CAR T cells were transduced to express eGFP.ffLuc. (A)FACS analysis confirmed the expression of the CAR and eGFP.ffLuctransgenes. (B, C) 1×10⁵ unmodified U373 cells were injectedintracranially into mice. On day 7, mice received 2×10⁶ 47.SSR.CD28.ζ.eGFP.ffLuc CAR T cells intracranially using the same tumorcoordinates. Bioluminescence imaging was used to monitor T cellpersistence.

FIG. 41A-C. Generation and characterization of LSR-CD28.41BB.ζ CAR Tcells. (A) Scheme of LSR.CD28.41BB.ζ CAR construct. (B) CAR expressionwas confirmed using FACS analysis. Representative plot. Open curve:secondary antibody; Filled curve: IL13Rα2Fc+ secondary antibody. (C)LSR.CD28.41BB.ζ CAR T cells were co-cultured with U373 cells at a 1:2E:T ratio. NT T cells served as controls. After 24 hours, IFNγ or IL2was measured by ELISA (n=3).

FIG. 42A-B. Generation of SSR.α.CD28.41BB.ζ CAR T cells. (A) Scheme ofSSR.α.CD28.41BB.ζ CAR construct. (B) CAR expression was tested usingFACS analysis (representative plot shown).

FIG. 43 . FACS analysis of PD-L1 expression on U373 cell surface withand without IFNγ stimulation. U373 cells were cultured with or withoutIFNγ (100 units/ml). After 24 hours, U373 cells were analyzed for PD-L1expression using a CD271 PE antibody (BD Biosciences).

FIG. 44A-B. Transgenic expression of IL15 in SSR.CD28.ζ T cells resultsin enhanced antigen-dependent IL15 secretion. T cells were stimulatedwith (A) recombinant proteins or (B) cell lines.

FIG. 45A-B. Transgenic expression of IL15 results in (A) enhanced invivo persistence of SSR.CD28.ζ T cells resulting in improved (B)progression-free survival (PFS).

DETAILED DESCRIPTION

The disclosure provides binding agents, or partners, that specificallyrecognize interleukin 13 receptor α2 (IL13Rα2) for use in diagnosing,preventing, treating or ameliorating a symptom of any of a wide range ofcancers characterized by cells presenting IL13Rα2. More particularly,the disclosure provides (i) the sequences of the six complementaritydetermining regions of a monoclonal antibody (m47) that specificallytargets human tumor-associated antigen, i.e., interleukin 13 receptor α2(IL13Rα2), and (ii) data demonstrating the functionality of the proteinencoded by the heavy and light chain cDNAs in the format of an scFvantibody or conjugate (e.g., fusion) to other functional moieties. Thesix complementarity determining regions of the m47 monoclonal antibodyconfer binding specificity for IL13Rα2, consistent with theunderstanding in the immunological arts. In some embodiments, the scFvcomprises the complete heavy and light chain variable regions ofantibody m47, or the complete heavy and light chains of antibody m47. Insome embodiments, the heavy and light chain fragments comprise, e.g.,the m47 CDRs, or the m47 variable regions, and these domains can bearranged in different formats, such as a single-chain variable fragmentof an antibody, i.e., a scFv, a diabody, a bi-specific antibodyfragment, a tri-specific antibody fragment, a fusion protein with any ofa wide variety of therapeutic proteins and/or other moieties, ahumanized antibody fragment, a Fab fragment, a Fab′ fragment, a F(ab)2′fragment and any other functional format for a bi-functional peptideproviding a targeting function and an effector function. Moreover, thesingle-chain antibody or other arrangements of the protein encoded bythe heavy and light chains could be expressed and conjugated totherapeutic carriers (e.g., viruses, cells, nanomaterials) for specificdelivery of a therapeutic to an IL13Rα2-expressing tumor. The materialsaccording to the disclosure are also useful in imaging tumor burden.

The technology addresses the most serious obstacle to progress inimmunotherapy, i.e., the virtual absence of defined, tumor-specificantigens that can be predictably found on at least a larger subgroup ofhuman cancers and that can serve as effective targets for cancereradication. Finding such antigens would move the field beyond themethods for treating CD19/CD20-expressing B cell malignancies.

The terms used throughout this disclosure are given their ordinary andaccustomed meanings in the art, unless a different meaning is made clearfrom the text when considered in the context of the disclosure as awhole.

The disclosure describes the development and characterization of amonoclonal antibody (mAb) fragment specific to IL13Rα2 for thetherapeutic purpose of targeting IL13Rα2-expressing tumors. The highaffinity IL13Rα2 is selectively expressed at a high frequency byglioblastoma multiforme (GBM) as well as several other tumor types. Oneapproach for targeting this tumor-specific receptor utilizes the cognateligand, IL-13, conjugated to cytotoxic molecules. This approach,however, lacks specificity because the lower affinity receptor forIL-13, IL13Rα1, is widely expressed by normal tissues. A monoclonalantibody (mAb) specific to IL13Rα2 was expected to overcome the lack ofspecificity afflicting methodologies that recognized both IL13receptors, i.e., IL13Rα1 as well as IL13Rα2. Such a mAb would betherapeutically useful in targeting and treating IL13Rα2-expressingcancers, including tumors.

As disclosed herein, hybridoma cell lines were generated and comparedfor binding affinities to recombinant human IL13Rα2 (rhIL13Rα2). Clone47 demonstrated binding to the native conformation of IL13Rα2 and wastherefore chosen for further studies. Clone 47 bound specifically andwith high affinity (KD=1.39×10⁻⁹M) to rhIL13Rα2 but not to rhIL13Rα1 ormurine IL13Rα2. Furthermore, clone 47 specifically recognized wild-typeIL13Rα2 expressed on the surface of CHO and HEK cells as well as severalglioma cell lines. Competitive binding assays revealed that clone 47also significantly inhibited the interaction between human soluble IL-13and IL13Rα2 receptor. Moreover, N-linked glycosylation of IL13Rα2contributes in part to the interaction of the antibody to IL13Rα2. Invivo, the IL13Rα2 mAb improved the survival of nude mice intracraniallyimplanted with a human U251 glioma xenograft.

The IL13Rα2-specific, scFv-based CAR, 47-CAR, constructed as disclosedherein, provided the material used in exploring the influence of longand short spacer regions, as well as endodomains, on its function. While47-CAR.SSR.CD28.ζ (i.e., the 47-CAR binding region provided as an scFvjoined to a short spacer region as defined herein, in turn joined to anunmodified or chimeric endodomain or T cell cytoplasmic domain) and47-CAR.LSR.CD28.ζ (similar construct substituting a long spacer region(LSR)) recognized target cells as judged by IFNγ production, only47-CAR.SSR.CD28.ζ induced IL2 production, indicating better T-cellactivation. An additional LSR 47-CAR containing a CD28.41BB.ζ endodomain(FIG. 41 ) was shown to lack the ability to induce IL2 expression. Theseobservations are consistent with knowledge that scFvs that bind to anepitope in close proximity to the cancer cell membrane, requiring longspacer regions for optimal CAR function, in contrast to scFvs that bindto epitopes distal to the cell membrane. The data disclosed hereinindicates that the IL13Rα2 epitope recognized by 47-CARs is locateddistal to the cell membrane.

In greater particularity, four SSR 47-CARs were constructed, each with adifferent endodomain, i.e., CD28.ζ, 41BB.ζ CD28.OX40.ζ, and CD28.41BB.ζ.While all four CARs were expressed, as judged by Western blot analysis,no significant cell-surface expression was observed for47-CAR.SSR.CD28.41BB.ζ. We explored if changing the transmembrane domainfrom CD28 to CD8a in 47-CAR.SSR.CD28.41BB.ζ would result in bettercell-surface expression, but no increase in expression was observed.Because 47-CARs.LSR.CD28.41BB.ζ are expressed on the cell surface (FIG.42 ), the result indicates that the interplay between spacer region andendodomain influences CAR cell-surface expression.

47-CAR.SSR.CD28.ζ 47-CAR.SSR.41BB.ζ, and 47-CAR.SSR.CD28.OX40.ζ T cellshad potent antitumor effect in vivo, resulting in a significant survivaladvantage. While mice treated with 47-CAR.SSR.CD28.ζ T cells had thelongest median survival in comparison to 47-CAR.SSR.41BB.ζ or47-CAR.SSR.CD28.OX40.ζ T-cell treated mice, this difference did notreach significance. The experimental results also showed that additionof a second costimulatory endodomain did not improve antitumor activityin vivo. Limited T-cell persistence in vivo was identified as theprincipal limitation on therapy. This limitation may be overcome by thetransgenic expression of cytokines³⁶ or by blocking inhibitory moleculesthat are secreted or present on the surface of gliomal cells. Forexample, gliomas such as U373 express PD-L1, which is upregulated in thepresence of IFNγ (FIG. 43 ), and could be targeted in future studies.

The experimental results disclosed herein establish that T cellsredirected to IL13Rα2 with 47-CARs have potent anti-tumor activityagainst glioma cells in vitro, and induce the regression of establishedGBM xenografts in vivo. 47-CARs are expected to be of value in thetreatment of not only IL13Rα2-positive GBMs but also other malignanciesin which IL13Rα2 is expressed.

The experimental results disclosed herein establish that T cellsredirected to IL13Rα2 with 47-CARs and that also express IL15 haveenhanced anti-tumor activity in the GBM xenografts in vivo.

The disclosure is based, at least in part, on the discovery that IL13Rα2is found preferentially on cancer cells such as tumor cells. Thisreceptor functions as a cancer-, or tumor-, specific antigen that hasbeen used to elicit the high-affinity monoclonal antibody m47, alongwith antigen binding fragments of that antibody. The VL and VH variableregions of the m47 antibody have been engineered into a single chain(sc) variable fragment (scFv) to generate conjugates, such as chimericantigen receptors (i.e., CARs), for introduction into T cells foradoptive transfer. Thus, CAR-transduced T cells are expected to target atumor-specific IL13Rα2 epitope, leading to eradication of cancer cellspresenting the receptor. It is believed that CAR-transduced T cellsrecognizing IL13Rα2 will destroy large solid tumors. CAR-transduced Tcells, however, target cancer cells only directly and antigen-negativecancer cells may escape. It is expected that CAR-transduced T cells alsowill be effective in eliminating antigen-negative cancer cells via thebystander effect.

Disclosed herein are experiments establishing the development ofIL13Rα2-specific CARs with a scFv47-based antigen-binding domain(47-CARs). The data show that 47-CARs perform better with a short spacerregion, which provides for optimal functionality, and that 47-CAR Tcells are able to recognize and kill only IL13Rα2-positive and notIL13Rα1-positive target cells in vitro. In addition, 47-CAR T cellsinduce tumor regression in an orthotopic xenograft mouse model of GBM,which was associated with a significant survival advantage.

The protein conjugates according to the disclosure are specific forIL13Rα2, which is associated with cancers, e.g., tumors. In addition,the disclosure provides a polynucleotide encoding one of thesecancer-specific binding partners, including polynucleotides comprisingcodon-optimized coding regions for binding partners specific for anepitope of IL13Rα2. The polynucleotides of the disclosure encodeconjugates, or bi-functional polypeptides, useful in diagnosing,preventing, treating, or ameliorating a symptom of cancer, such as anyof a variety of human cancers, including those forming solid tumors.Also contemplated are vectors comprising a polynucleotide as disclosedherein, a host cell comprising such a polynucleotide and/or a vector asdescribed above, and methods of treating, preventing or ameliorating asymptom of, a cancer disease, e.g., a solid tumor, a primary cancer siteor a metastasized cancer.

The various forms of conjugates known in the art are contemplated by thedisclosure. These conjugates provide exquisitely cancer- as well asprotein-specific antibody receptors that can be incorporated into avariety of backbones providing effector function, such as bispecific Tcell Engagers (BiTEs) or chimeric antigen receptors (CARs), as notedbelow. Exemplary conjugates of the disclosure include CARs, fusionproteins, including fusions comprising single-chain variable (antibody)fragment (scFv) multimers or scFv fusions to coding regions encodingproducts useful in treating cancer, e.g., IL15, IL15Rα, or IL15/IL15Rαagent, diabodies, tribodies, tetrabodies, and bispecific bivalent scFvs,including bispecific tandem bivalent scFvs, also known as bispecific Tcell engagers, or BiTEs. Any of these conjugate forms, moreover, mayexhibit any of various relative structures, as it is known in the artthat different domain orders (e.g., H₂N-VH-linker-VL-CO₂H andH₂N-VL-linker-VH-CO₂H) are compatible with specific binding. Higherorder forms of the conjugates described herein are also contemplated,such as peptibodies comprising at least one form of the conjugatesdisclosed herein. The conjugates of the disclosure specifically bind toa cancer-specific epitope (e.g., an IL13Rα2) and the polynucleotidesencoding them may be codon-optimized, e.g., for maximal translation, forexpression in the targeted cells (e.g., human or mouse cells). Codonoptimization in the context of expressing the conjugates of thedisclosure, such as CARs, is important to ensuring that production ofthe protein is both efficient and robust enough to be useful as a sourceof therapeutic.

The disclosure also contemplates conjugates in which a targeting moiety(an anti-IL13Rα2 antibody or fragment thereof) is linked to a peptideproviding a second function, e.g., an effector function, such as a Tcell signaling domain involved in T cell activation, a peptide thataffects or modulates an immunological response to cancer cells, or anenzymatic component of a labeling system that results in a CAR encodedby a polynucleotide according to the disclosure, if the coding regionfor the conjugate is codon-optimized for expression in a target cell.Exemplary conjugates include an anti-IL13Rα2 scFv linked to a hinge, atransmembrane domain, and an effector compound or domain, e.g., CD28,CD3ζ, CD134 (OX40), CD137 (41BB), ICOS, CD40, CD27, or Myd88, therebyyielding a CAR.

The polynucleotide aspect of the disclosure comprises embodiments inwhich an unexpected variation on codon optimization in slower-growinghigher eukaryotes such as vertebrates, e.g., humans, is provided that isfocused on translation optimization (maximizing high-fidelitytranslation rates) rather than the typical codon optimization used insuch organisms, which is designed to accommodate mutational bias andthereby minimize mutation. Also disclosed are the methods of diagnosing,preventing, treating or ameliorating a symptom of a cancer.Schematically described, the polynucleotides comprise a codon-optimizedcoding region for an antigen receptor specifically recognizing anIL13Rα2 epitope linked to any one of the following: a coding region fora T cell signaling domain involved in T cell activation, a gene productthat affects or modulates an immunological response to cancer cells suchas an IL15/IL15Rα fusion, or a labeling component such as an enzymaticcomponent of a labeling system. The linked coding regions result inpolynucleotides encoding conjugates according to the disclosure, such asBiTEs or chimeric antigen receptors (CARs).

In methods of diagnosing, preventing, treating or ameliorating a symptomof a cancer, the compositions of the disclosure are typicallyadministered in the form of a conjugate-transduced cell, such as a Tcell, an NK cell, or a lymphocyte including, but not limited to, NKTcells, γδ T cells, mucosa associated invariant T cells or MAIT cells, orinnate lymphoid cells, although administration of a vector comprising apolynucleotide of the disclosure or administration of a polynucleotideof the disclosure are also contemplated, depending on thefunctionalities of the conjugate. Combining a polynucleotide, vector orhost cell of the disclosure with a physiologically suitable buffer,adjuvant or diluent yields a pharmaceutical composition according to thedisclosure, and these pharmaceutical compositions are suitable foradministration to diagnose, prevent, treat, or ameliorate a symptom of,a cancer.

In the course of experimental work described herein, hybridoma celllines were generated and compared for binding affinities to recombinanthuman IL13Rα2 (rhIL13Rα2). Clone 47 demonstrated binding to the nativeconformation of IL13Rα2 and was therefore characterized further. Clone47 bound specifically and with high affinity (KD 1.39×10⁻⁹ M) torhIL13Rα2 but not to rhIL13Rα1 or murine IL13Rα2. Furthermore, clone 47specifically recognized wild-type IL13Rα2 expressed on the surface ofCHO and HEK cells as well as several glioma cell lines. Competitivebinding assays revealed that clone 47 also significantly inhibited theinteraction between human soluble IL-13 and IL13Rα2 receptor. Moreover,N-linked glycosylation of IL13Rα2 was found to contribute, in part, tothe interaction of the antibody with IL13Rα2. In vivo, the IL13Rα2monoclonal antibody improved the survival of nude mice intracraniallyimplanted with a human U251 glioma xenograft. Collectively, these dataestablish the efficacy of the immunomodulatory treatment of cancerdisclosed herein.

Overexpression of IL13Rα2 in glioblastoma multiforme (GBM) but not innormal brain tissue uniquely positions this receptor as a candidate fortargeting tumor cells. GBM is a highly infiltrative tumor, often makingcomplete surgical removal impossible. Moreover, GBM is highly resistantto radiation and chemotherapy (16), warranting further development ofnovel and targeted therapies for the treatment of patients.

A phage display library approach has been used to select small antibodyfragments specific to human IL13Rα2, followed by their evaluation invitro and in vivo (23). Despite the high specificity of interaction withIL13Rα2, conjugation with toxins has failed to increase cytotoxicity inIL13Rα2-expressing glioma and renal cell carcinoma cell lines whencompared with the effects of IL-13PE38. The low affinity of generatedantibody fragments is the most reasonable explanation for the lack ofsuccess. Antibody fragments derived from phage display libraries areknown to be lower in affinity and avidity than antibodies generated byconventional hybridoma technology (24). Modifications of those smallantibody fragments are often required to enhance their affinity andavidity to targeted proteins. In recent years, monoclonal antibodieshave shown increasing success as targeted anticancer and diagnosticagents (25, 26), and a further search for high affinity reagents withrestricted specificity to tumor-associated antigens is needed. Theexperiments disclosed herein were designed to discover, develop, andcharacterize a high affinity antibody that specifically recognizesIL13Rα2 expressed on the surface of cancer cells. Consistent with thatdesign, disclosed herein are experiments establishing the generation ofan antibody possessing the properties critical for immunotherapeutictargeting of IL13Rα2-expressing tumors in vivo, and potentially suitablefor various other applications.

Monoclonal antibodies appear to be valuable research and diagnostictools as well as therapeutic agents. Monoclonal antibodies specific fortumor-associated antigens have significant advantages over systemicchemotherapies due to the ability to specifically target cancer cellswhile avoiding interaction with untransformed tissue. Therefore, thesearch for novel “magic bullets” continues to grow, confirmed by aglobal market for therapeutic antibodies worth $48 billion as of 2010.Therapeutic antibodies are products of traditional hybridoma technologyor screening of libraries for antibody fragments and their subsequentengineering into humanized fragments or full size molecules. Prior tothis study, the hybridoma cell line secreting a high affinity antibodyto the tumor-specific antigen IL13Rα2 was unavailable to the scientificcommunity. Here, we describe the generation and characterization of ahigh affinity antibody to the tumor-specific antigen IL13Rα2 and discussits potential use in different applications.

The specificity of interaction of newly discovered antibodies to humanIL13Rα2 was analyzed by ELISA using the rhIL13Rα2hFc fusion protein,recombinant human IL13Rα2 expressed on the surface of CHO and HEK cells,and several glioma cell lines expressing IL13Rα2 at various levels byflow cytometry. The antibody identified herein, and agent using thebinding domain thereof, demonstrated a specificity of interaction tohuman IL13Rα2 and did not cross-react with human IL13Rα1 or mouseIL13Rα2. Moreover, the specificity of binding to IL13Rα2 was confirmedin competitive binding assays using rhIL13Rα2hFc fusion protein by ELISAor by flow cytometry for detection of IL13Rα2 expressed on the surfaceof HEK cells. In these assays, IL13Rα2 (clone 47) mAb competed withrecombinant human IL-13 for its epitope and was able to block about 80%of the binding between IL-13 and IL13Rα2. Conversely, human recombinantIL-13 was able to block about 50% of antibody binding to IL13Rα2.Similarly, a significant decrease in the binding of IL13Rα2 (clone 47)mAb to N10 glioma cells was observed when rhIL13R2hFc chimera andrhIL-13 were used as competitors. The binding of rhIL-13 to N10 cellswas also abolished by IL13Rα2 (clone 47) mAb. These data indicate thatthe two molecules have significant overlap in their recognition sitesfor IL13Rα2.

IL-13 is a small 10-kDa molecule (31), whereas an antibody is about 15times greater in molecular mass. The ability of rhIL-13 to compete withan antibody for a binding site suggests that the inhibitory property ofthe antibody is likely due to the specific interaction with amino acidresidues contributing to the binding of IL-13 to the cognate receptorrather than to steric hindrance, which can also prevent the interactionof IL-13 with its receptor. Previously, Tyr207, Asp271, Tyr315, andAsp318 were identified as critical residues of IL13Rα2 necessary forinteraction with IL-13 (28). In the assays disclosed herein, the bindingof IL-13 to a mutant IL13Rα2 carrying a combination of all 4 amino acidmutations to alanine was significantly abolished when compared with thewild-type receptor. Binding of the IL13Rα2 mAb to either the individualor the 4-amino acid mutant form of IL13Rα2, however, was notsignificantly affected. These findings indicate that Tyr207, Asp271,Tyr315, and Asp318 residues are not critical for the recognition ofIL13Rα2 by the IL13Rα2 mAb. The human IL13Rα2 and murine IL13Rα2 arestructurally conserved and share 59% amino acid identity (32). Moreover,Tyr207, Asp271, Tyr315, and Asp318 residues are conserved in human andmurine IL13Rα2. Absence of binding of the IL13Rα2 mAb to murineIL13Rα2hFc fusion further supports the expectation that these amino acidresidues contribute to the binding of IL-13 to IL13Rα2 and are notcritical for the interaction of this antibody with the receptor.

To further characterize the interaction of IL13Rα2 with the antibody andantibody agent disclosed herein, the affinity of the IL13Rα2 mAb wasmeasured and compared with the binding properties of two commerciallyavailable antibodies using the surface plasmon resonance method. Theaffinity of the IL13Rα2 mAb was determined to be equal to 1.39×10⁻⁹M,greatly exceeding the affinity of comparable commercially availableantibodies by up to 75-fold. In agreement with the affinity studies, theIL13Rα2 mAb (clone 47) demonstrated superiority to two commercialantibodies in binding to the IL13Rα2 expressed on the surface of variousglioma cells and in ELISA. Although many properties of antibodies,including the affinity and avidity, in vivo stability, rate of clearanceand internalization, tumor penetration, and retention, should beconsidered prior to specific usage, it has been reported that higheraffinity antibodies are better for immunotherapeutic tumor-targetingapplications (33). The single chain antibody fragment (scFv) MR1-1against epidermal growth factor receptor variant III demonstrates about15-fold higher affinity than the parental scFvMR1 and also showed onaverage a 244% higher tumor uptake than that for the scFvMR1 (34). It islikely that the high affinity properties of the IL13Rα2 mAb and agentthereof that are disclosed herein will be advantageous for applicationsutilizing antibodies or associated derivatives for targeting tumor cellsexpressing IL13Rα2.

The N-linked glycosylation of IL13Rα2 has been identified as a necessaryrequirement for efficient binding to IL-13 (30). Taking intoconsideration that the IL13Rα2 mAb disclosed herein inhibits about 80%of IL-13 binding to the cognate receptor, IL13Rα2, it is reasonable toexpect that the binding of this antibody, or an agent containing itsbinding domain, with the deglycosylated form of IL13Rα2 could also beaffected. The IL13Rα2 molecule has four potential sites of N-linkedglycosylation. The binding of the antibody to rhIL13Rα2 or to IL13Rα2expressed on the surface of HEK or U251 cells treated with Pngase F wasdecreased by 35 and 30%, respectively, when compared with non-treatedcontrol. A partial change in binding activity for the clone 47 whencompared with clones 83807 and B-D13 suggests that removal ofcarbohydrate adducts from IL13Rα2 with Pngase F causes conformationalchanges of the receptor, indirectly affecting the binding of both IL-13(30) and the IL13Rα2 mAb to IL13Rα2. This also supports the expectationthat the antibody binds directly to the IL13Rα2 amino acid backbonerather than interacting with carbohydrate moieties addedpost-translationally. Supporting this expectation, several studies havepreviously demonstrated that the conformational profile and structuralrigidity of proteins depends on N-linked glycosylation (22, 35-38).

To investigate the therapeutic properties of the IL13Rα2 mAb and itsagent, an in vivo study was performed whereby glioma cells and theIL13Rα2 (clone 47) mAb were intracranially co-injected into brain, orantibody was injected into established tumor-bearing mice.Interestingly, the IL13Rα2 mAb was able to delay tumor progression andimprove survival of animals with intracranial U251 glioma xenograftsmost significantly in the co-injected model, demonstrating a trend inthe improvement of median survival in animals with established glioma.Although the underlying mechanism for this antitumor effect remainsunclear, the result establishes the therapeutic applicability of thisantibody, or its agent (containing the IL13Rα2 binding domain in theform of the six CDR regions, or in the form of the two variable domainsof the clone 47 anti-IL13Rα2), alone or in combination with apharmaceutical carrier, thereby providing therapies for the treatment ofIL13Rα2-expressing glial and other lineage tumors. Several antibodieshave been shown to mediate a cytotoxic effect in tumors throughFc-mediated activation of complement (39). Antibody-dependentcell-mediated cytotoxicity-induced activation of effector cells can alsocontribute to the cytotoxic effect of antibodies against targeted cells(40, 41). Anti-IL13Rα2 activity derived from the sera of animalschallenged with D5 melanoma cells expressing human IL13Rα2 demonstratesthe ability to inhibit cellular growth in vitro (4).

Cancers amenable to the described treatments include cancers in whichIL13Rα2 has been found to be expressed, including glioblastoma;medulloblastoma; Kaposi sarcoma; and head and neck, ovarian, pancreatic,kidney, and colorectal cancers (2, 43-47). Although the role of IL13Rα2in some cancers is not yet defined, recent reports have demonstratedthat IL13Rα2 contributes to the invasive phenotype of ovarian,pancreatic, and colorectal cancers (5, 13). Moreover, Minn et al. (42)have suggested a relationship between IL13Rα2 expression and breastcancer metastasis to the lung. Additionally, Fichtner-Feigl et al. (11)demonstrated that the interaction of IL-13 with IL13Rα2 upregulatesTGF-β1, mediating fibrosis in a bleomycin-induced model of lungfibrosis. In light of this finding, it is expected that the anti-IL13Rα2antibody (clone 47) and binding agents thereof, will be able toattenuate TGF-β1-induced pulmonary fibrosis.

As disclosed herein, the described experiments led to the generation ofan anti-IL13Rα2 antibody and binding agents thereof, all of which arespecific to human IL13Rα2. The antibody and its agent possess a highaffinity for IL13Rα2 and compete with IL-13 for the binding site onIL13Rα2. The antibody recognizes antigen expressed on the cell surfaceof glioma cells as well as other IL13Rα2-expressing cells, establishingthe suitability for targeting IL13Rα2-expressing tumor cells in vivo.The anti-IL13Rα2 antibody and binding agents thereof are also expectedto be efficacious and cost effective in diagnostic imaging, delivery ofantibody radionuclide conjugates, bioassays for the detection ofIL13Rα2, and as a carrier for therapeutic agents in various types ofIL13Rα2-overexpressing tumors.

In methods of diagnosing, preventing, treating or ameliorating a symptomof a cancer, the compositions of the disclosure are typicallyadministered in the form of conjugate-transduced T cells, althoughadministration of a vector comprising a polynucleotide of the disclosureor administration of a polynucleotide of the disclosure are alsocontemplated, depending on the functionalities of the conjugate.Combining a polynucleotide, vector or host cell of the disclosure with aphysiologically suitable buffer, adjuvant or diluent yields apharmaceutical composition according to the disclosure, and thesepharmaceutical compositions are suitable for administration to diagnose,prevent, treat, or ameliorate a symptom of, a cancer.

A conjugate according to the disclosure, such as a fusion proteincomposed of an scFv-receptor for an IL13Rα2 epitope fused toIL15/IL15Rα, is also contemplated. It is expected that the fusionprotein will eliminate clinical size tumors or only incipient andmicrodisseminated cancer cells. The disclosure further contemplates thesimultaneous targeting of two independent IL13Rα2 epitopes on a humancancer, which may be essential for preventing escape from treatment,such as CAR treatment.

Simultaneous targeting of different epitopes of IL13Rα2 by CARs shouldreduce the chance of escape of a cancer subpopulation, which provides astrong reason for identifying additional IL13Rα2 antibody productsand/or epitopes.

The disclosure provides materials and methods that are adaptable and canserve as the basis for a platform technology with considerable growthpotential. The cancer-specific nature of IL13Rα2 is expected to providetargets for cancer diagnostics, prophylactics and therapeutics thatoffer major advantages over previously and presently used targets.

Consistent with the spirit of the foregoing, the following provides adescription of the materials and methods provided herein.

Disclosed herein are IL13Rα2 binding agents comprising each of the aminoacid sequences of NYLMN (SEQ ID NO: 1); RIDPYDGDIDYNQNFKD (SEQ ID NO:2); GYGTAYGVDY (SEQ ID NO: 3); RASESVDNYGISFMN (SEQ ID NO: 4); AASRQGSG(SEQ ID NO: 5); and QQSKEVPWT (SEQ ID NO: 6). In exemplary aspects, thebinding agent comprises each of the foregoing six amino acid sequencesin addition to further sequences which provide a framework to support athree-dimensional conformation that binds to IL13Rα2. In exemplaryaspects, the IL13Rα2 binding agent comprises one or both of the aminoacid sequences of SEQ ID NO: 7 and/or SEQ ID NO: 8. In exemplaryaspects, the IL13Rα2 binding agent comprises the amino acid sequence ofSEQ ID NO: 7. In exemplary aspects, the IL13Rα2 binding agent comprisesthe amino acid sequence of SEQ ID NO: 8. In exemplary aspects, theIL13Rα2 binding agent comprises both the amino acid sequences of SEQ IDNO: 7 and SEQ ID NO: 8. In exemplary aspects wherein both the amino acidsequences of SEQ ID NO: 7 and SEQ ID NO: 8 are present in the bindingagent, the amino acid sequence of SEQ ID NO: 7 is fused to the aminoacid sequence of SEQ ID NO: 8 through a linker. Suitable linkers areknown in the art. In exemplary aspects, the linker comprises a shortamino acid sequence of about 5 to about 25 amino acids, e.g., about 10to about 20 amino acids. In exemplary aspects, the linker comprises theamino acid sequence of EEGEFSEAR (SEQ ID NO 10). In exemplary aspects,the linker comprises the amino acid sequence of AKTTPPKLEEGEFSEARV (SEQID NO: 80). In exemplary aspects, IL13Rα2 binding agent comprises theamino acid sequence of SEQ ID NO: 13.

In exemplary embodiments, the binding agent provided herein furthercomprises additional amino acid sequences. In exemplary aspects, thebinding agent further comprises a constant region of a heavy chainand/or a constant region of a light chain. Sequences for heavy and lightchain constant regions are publically available. For example, theNational Center of Biotechnology Information (NCBI) nucleotide databaseprovides a sequence of the constant region of the IgG1 kappa lightchain. See GenBank Accession No. DQ381549.1, incorporated herein byreference. In exemplary aspects, the binding agent comprises an aminoacid sequence of SEQ ID NO: 28. In exemplary aspects, the binding agentcomprises a modified amino acid sequence of SEQ ID NO: 28. In exemplaryaspects, the binding agent comprises an amino acid sequence which is atleast 90%, at least 93%, at least 95%, or at least 98% identical to SEQID NO: 28. Also, for example, the NCBI nucleotide database provides asequence of the constant region of the Mus musculus IgG1. See GenBankAccession No. DQ381544.1. In exemplary aspects, the binding agentcomprises an amino acid sequence of SEQ ID NO: 29. In exemplary aspects,the binding agent comprises a modified amino acid sequence of SEQ ID NO:29. In exemplary aspects, the binding agent comprises an amino acidsequence which is at least 90%, at least 93%, at least 95%, or at least98% identical to SEQ ID NO: 29.

In exemplary aspects, the IL13Rα2 binding agent is an antibody, or anantigen-binding fragment thereof. In exemplary aspects, the antibodycomprises each of the amino acid sequences of SEQ ID NOs: 1-6. Inexemplary aspects, the antibody comprises the amino acid sequence of SEQID NO: 7 and/or SEQ ID NO: 8. In exemplary aspects, the antibodycomprises the amino acid sequences of SEQ ID NO: 7 and SEQ ID NO: 8. Inexemplary aspects, the antibody comprises the amino acid sequences ofSEQ ID NO: 7 and SEQ ID NO: 8 and the amino acid sequence of SEQ ID NO:7 is fused to the amino acid sequence of SEQ ID NO: 8 through a linker.In exemplary aspects, the linker comprises a short amino acid sequenceof about 5 to about 25 amino acids, e.g., about 10 to about 20 aminoacids. In exemplary aspects, the linker comprises the amino acidsequence of EEGEFSEAR (SEQ ID NO 10). In exemplary aspects, the linkercomprises the amino acid sequence of AKTTPPKLEEGEFSEARV (SEQ ID NO: 80).In exemplary aspects, the antibody comprises the amino acid sequence ofSEQ ID NO: 13.

In exemplary aspects, the antibody can be any type of immunoglobulinthat is known in the art. For instance, the antibody can be of anyisotype, e.g., IgA, IgD, IgE, IgG, or IgM. The antibody can bemonoclonal or polyclonal. The antibody can be a naturally-occurringantibody, i.e., an antibody isolated and/or purified from a mammal,e.g., mouse, rabbit, goat, horse, chicken, hamster, human, and the like.In this regard, the antibody may be considered to be a mammalianantibody, e.g., a mouse antibody, rabbit antibody, goat antibody, horseantibody, chicken antibody, hamster antibody, human antibody, and thelike. The term “isolated” as used herein means having been removed fromits natural environment. The term “purified,” as used herein relates tothe isolation of a molecule or compound in a form that is substantiallyfree of contaminants normally associated with the molecule or compoundin a native or natural environment and means having been increased inpurity as a result of being separated from other components of theoriginal composition. It is recognized that “purity” is a relative term,and not to be necessarily construed as absolute purity or absoluteenrichment or absolute selection. In some aspects, the purity is atleast or about 50%, is at least or about 60%, at least or about 70%, atleast or about 80%, or at least or about 90% (e.g., at least or about91%, at least or about 92%, at least or about 93%, at least or about94%, at least or about 95%, at least or about 96%, at least or about97%, at least or about 98%, at least or about 99% or is approximately100%.

In exemplary aspects, the antibody comprises a constant region of anIgG. In exemplary aspects, the antibody comprises a constant region ofan IgG₁. In exemplary aspects, the antibody comprises a constant regionof an IgG kappa light chain. For instance, the antibody may comprise theamino acid sequence of SEQ ID NO: 28. In exemplary aspects, the antibodycomprises an amino acid sequence that is highly similar to SEQ ID NO:28. For instance, the antibody may comprise an amino acid sequencehaving at least 85% sequence identity to SEQ ID NO: 28, or an amino acidsequence having at least 90% sequence identity to SEQ ID NO: 28, or anamino acid sequence having at least 93% sequence identity to SEQ ID NO:28, or an amino acid sequence having at least 95% sequence identity toSEQ ID NO: 28, or an amino acid sequence having at least 98% sequenceidentity to SEQ ID NO: 28.

In exemplary aspects, the antibody comprises a constant region of a Musmusculus IgG₁. For instance, the antibody may comprise the amino acidsequence of SEQ ID NO: 30. In exemplary aspects, the antibody comprisesan amino acid sequence which is highly similar to SEQ ID NO: 30. Forinstance, the antibody may comprise an amino acid sequence having atleast 85% sequence identity to SEQ ID NO: 30, or an amino acid sequencehaving at least 90% sequence identity to SEQ ID NO: 30, or an amino acidsequence having at least 93% sequence identity to SEQ ID NO: 30, or anamino acid sequence having at least 95% sequence identity to SEQ ID NO:30, or an amino acid sequence having at least 98% sequence identity toSEQ ID NO: 30.

The anti-IL13Rα2 antibodies and fragments thereof of the disclosure canhave any level of affinity or avidity for IL13Rα2. The dissociationconstant (K_(D)) may be any of those exemplary dissociation constantsdescribed herein with regard to binding units. Binding constants,including dissociation constants, are determined by methods known in theart, including, for example, methods that utilize the principles ofsurface plasmon resonance, e.g., methods utilizing a Biacore™ system. Inaccordance with the foregoing, in some embodiments, the antibody is inmonomeric form, while in other embodiments, the antibody is in polymericform. In certain embodiments in which the antibody comprises two or moredistinct antigen binding regions or fragments, the antibody isconsidered bispecific, trispecific, or multi-specific, or bivalent,trivalent, or multivalent, depending on the number of distinct epitopesthat are recognized and bound by the binding agent.

Because the binding agent of the disclosures can compete with IL13 forbinding to IL13Rα2, the antibody in exemplary aspects is considered tobe a blocking antibody or neutralizing antibody. In some aspects, theK_(D) of the binding agent is about the same as the K_(D) of the nativeligand, IL13, for IL13Rα2. In some aspects, the K_(D) of the bindingagent is lower (e.g., at least 0.5-fold lower, at least 1-fold lower, atleast 2-fold lower, at least 5-fold lower, at least 10-fold lower, atleast 25-fold lower, at least 50-fold lower, at least 75-fold lower, atleast 100-fold lower) than the K_(D) of IL13 for IL13Rα2. In exemplaryaspects, the K_(D) is between about 0.0001 nM and about 100 nM. In someembodiments, the K_(D) is at least or about 0.0001 nM, at least or about0.001 nM, at least or about 0.01 nM, at least or about 0.1 nM, at leastor about 1 nM, or at least or about 10 nM. In some embodiments, theK_(D) is no more than or about 100 nM, no more than or about 75 nM, nomore than or about 50 nM, or no more than or about 25 nM. In exemplaryaspects, the antibody has a K_(D) for human IL13Rα2 that is no greaterthan about 1.39×10⁻⁹M.

In exemplary aspects, the binding agent, e.g., antibody, or antigenbinding fragment thereof, does not bind to human IL13Rα1.

In exemplary embodiments, the antibody is a genetically engineeredantibody, e.g., a single chain antibody, a humanized antibody, achimeric antibody, a CDR-grafted antibody, an antibody that includesportions of CDR sequences specific for IL13Rα2 (e.g., an antibody thatincludes CDR sequences of SEQ ID NOs: 1-6), a humaneered or humanizedantibody, a bispecific antibody, a trispecific antibody, and the like,as defined in greater detail herein. Genetic engineering techniques alsoprovide the ability to make fully human antibodies in a non-human.

In some aspects, the antibody is a chimeric antibody. The term “chimericantibody” is used herein to refer to an antibody containing constantdomains from one species and the variable domains from a second, or moregenerally, containing stretches of amino acid sequence from at least twospecies.

In some aspects, the antibody is a humanized antibody. The term“humanized” when used in relation to antibodies is used to refer toantibodies having at least CDR regions from a nonhuman source that areengineered to have a structure and immunological function more similarto true human antibodies than the original source antibodies. Forexample, humanizing can involve grafting CDR from a non-human antibody,such as a mouse antibody, into a human antibody. Humanizing also caninvolve select amino acid substitutions to make a non-human sequencelook more like a human sequence, as would be known in the art.

Use of the terms “chimeric or humanized” herein is not meant to bemutually exclusive; rather, is meant to encompass chimeric antibodies,humanized antibodies, and chimeric antibodies that have been furtherhumanized. Except where context otherwise indicates, statements about(properties of, uses of, testing, and so on) chimeric antibodies applyto humanized antibodies, and statements about humanized antibodiespertain also to chimeric antibodies. Likewise, except where contextdictates, such statements also should be understood to be applicable toantibodies and antigen binding fragments of such antibodies.

In some aspects of the disclosure, the binding agent is an antigenbinding fragment of an antibody that specifically binds to an IL13Rα2 inaccordance with the disclosure. The antigen binding fragment (alsoreferred to herein as “antigen binding portion”) may be an antigenbinding fragment of any of the antibodies described herein. The antigenbinding fragment can be any part of an antibody that has at least oneantigen binding site, including, but not limited to, Fab, F(ab′)₂, dsFv,sFv, scFv, diabodies, triabodies, bis-scFvs, fragments expressed by aFab expression library, domain antibodies, VhH domains, V-NAR domains,VH domains, VL domains, and the like. Antibody fragments of theinvention, however, are not limited to these exemplary types of antibodyfragments.

In exemplary aspects, the IL13Rα2 binding agent is an antigen bindingfragment. In exemplary aspects, the antigen binding fragment compriseseach of the amino acid sequences of SEQ ID NOs: 1-6. In exemplaryaspects, the antigen binding fragment comprises the amino acid sequenceof SEQ ID NO: 7 and/or SEQ ID NO: 8. In exemplary aspects, the antigenbinding fragment comprises the amino acid sequences of SEQ ID NO: 7 andSEQ ID NO: 8. In exemplary aspects, the antigen binding fragmentcomprises the amino acid sequences of SEQ ID NO: 7 and SEQ ID NO: 8 andthe amino acid sequence of SEQ ID NO: 7 is fused to the amino acidsequence of SEQ ID NO: 8 through a linker. In exemplary aspects, thelinker comprises a short amino acid sequence of about 5 to about 25amino acids, e.g., about 10 to about 20 amino acids. In exemplaryaspects, the linker comprises the amino acid sequence of EEGEFSEAR (SEQID NO 10). In exemplary aspects, the linker comprises the amino acidsequence of AKTTPPKLEEGEFSEARV (SEQ ID NO: 80). In exemplary aspects,the antigen binding fragment provided herein comprises the amino acidsequence of SEQ ID NO: 13.

In exemplary aspects, the antigen binding fragment comprises a leadersequence. Optionally, the leader sequence, in some aspects, is locatedN-terminal to the heavy chain variable region. In exemplary aspects, theantigen binding fragment comprises an Ig kappa leader sequence. Suitableleader sequences are known in the art, and include, for example, an Igkappa leader sequence of METDTLLLWVLLLWVPGSTGD (SEQ ID NO: 9).

In exemplary aspects, an antigen binding fragment comprises one more tagsequences. Tag sequences may assist in the production andcharacterization of the manufactured antigen binding fragment. Inexemplary aspects, the antigen binding fragment comprises one or moretag sequences C-terminal to the light chain variable region. Suitabletag sequences are known in the art and include, but are not limited to,Myc tags, His tags, and the like. In exemplary aspects, an antigenbinding fragment comprises a Myc tag of GGPEQKLISEEDLN (SEQ ID NO: 11).In exemplary aspects, an antigen binding fragment comprises a His tagsequence of HHHHHH (SEQ ID NO: 12).

In exemplary aspects, the antigen binding fragment of the disclosurescomprises, from the N- to the C-terminus, a leader sequence, a heavychain variable region, a linker sequence, a light chain variable region,a Myc tag (e.g., SEQ ID NO: 11), and a His tag (e.g., SEQ ID NO: 12). Inexemplary aspects, the antigen binding fragment of the disclosurecomprises the amino acid sequence of SEQ ID NO: 14.

In exemplary aspects, the antigen binding fragment is a domain antibody.A domain antibody comprises a functional binding unit of an antibody,and can correspond to the variable regions of either the heavy (V_(H))or light (V_(L)) chains of antibodies. A domain antibody can have amolecular weight of approximately 13 kDa, or approximately one-tenth theweight of a full antibody. Domain antibodies may be derived from fullantibodies, such as those described herein. The antigen bindingfragments in some embodiments are monomeric or polymeric, bispecific ortrispecific, and bivalent or trivalent.

Antibody fragments that contain the antigen binding, or idiotope, of theantibody molecule share a common idiotype and are contemplated by thedisclosure. Such antibody fragments may be generated by techniques knownin the art and include, but are not limited to, the F(ab′)₂ fragmentwhich may be produced by pepsin digestion of the antibody molecule; theFab′ fragments which may be generated by reducing the disulfide bridgesof the F(ab′)₂ fragment, and the two Fab′ fragments which may begenerated by treating the antibody molecule with papain and a reducingagent.

In exemplary aspects, the binding agent provided herein is asingle-chain variable region fragment (scFv) antibody fragment. An scFvmay consist of a truncated Fab fragment comprising the variable (V)domain of an antibody heavy chain linked to a V domain of an antibodylight chain via a synthetic peptide, and it can be generated usingroutine recombinant DNA technology techniques (see, e.g., Janeway etal., Immunobiology, 2^(nd) Edition, Garland Publishing, New York,(1996)). Similarly, disulfide-stabilized variable region fragments(dsFv) can be prepared by recombinant DNA technology (see, e.g., Reiteret al., Protein Engineering, 7, 697-704 (1994)).

In exemplary aspects, the IL13Rα2 binding agent provided herein is anscFv. In exemplary aspects, the scFv comprises each of the amino acidsequences of SEQ ID NOs: 1-6. In exemplary aspects, the scFv comprisesthe amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 8. In exemplaryaspects, the scFv comprises the amino acid sequences of SEQ ID NO: 7 andSEQ ID NO: 8. In exemplary aspects, the scFv comprises the amino acidsequences of SEQ ID NO: 7 and SEQ ID NO: 8 and the amino acid sequenceof SEQ ID NO: 7 is fused to the amino acid sequence of SEQ ID NO: 8through a linker. In exemplary aspects, the linker comprises a shortamino acid sequence of about 5 to about 25 amino acids, e.g., about 10to about 20 amino acids. In exemplary aspects, the linker comprises theamino acid sequence of EEGEFSEAR (SEQ ID NO 10). In exemplary aspects,the linker comprises the amino acid sequence of AKTTPPKLEEGEFSEARV (SEQID NO: 80). In exemplary aspects, the scFv provided herein comprises theamino acid sequence of SEQ ID NO: 13.

Recombinant antibody fragments, e.g., scFvs of the disclosure, can alsobe engineered to assemble into stable multimeric oligomers of highbinding avidity and specificity to different target antigens. Suchdiabodies (dimers), triabodies (trimers) or tetrabodies (tetramers) arewell known in the art. See e.g., Kortt et al., Biomol Eng. 200118:95-108, (2001) and Todorovska et al., J Immunol Methods. 248:47-66,(2001).

In exemplary aspects, the binding agent is a bispecific antibody(bscAb). Bispecific antibodies are molecules comprising two single-chainFv fragments joined via a glycine-serine linker using recombinantmethods. The V light-chain (V_(L)) and V heavy-chain (V_(H)) domains oftwo antibodies of interest in exemplary embodiments are isolated usingstandard PCR methods. The V_(L) and V_(H) cDNAs obtained from eachhybridoma are then joined to form a single-chain fragment in a two-stepfusion PCR. Bispecific fusion proteins are prepared in a similar manner.Bispecific single-chain antibodies and bispecific fusion proteins areantibody substances included within the scope of the present invention.Exemplary bispecific antibodies are taught in U.S. Patent ApplicationPublication No. 2005-0282233A1 and International Patent ApplicationPublication No. WO 2005/087812, both applications of which areincorporated herein by reference in their entireties.

In exemplary aspects, the binding agent is a bispecific T-cell engagingantibody (BiTE) containing two scFvs produced as a single polypeptidechain. In exemplary aspects, the binding agent is a BiTE comprising twoscFvs, wherein at least one comprises each of the amino acid sequencesof SEQ ID NOs: 1-6 or comprises SEQ ID NO: 7 and/or SEQ ID NO: 8.Methods of making and using BiTE antibodies are described in the art.See, e.g., Cioffi et al., Clin Cancer Res 18: 465, Brischwein et al.,Mol Immunol 43:1129-43 (2006); Amann M et al., Cancer Res 68:143-51(2008); Schlereth et al., Cancer Res 65: 2882-2889 (2005); and Schlerethet al., Cancer Immunol Immunother 55:785-796 (2006).

In exemplary aspects, the binding agent is a dual affinity re-targetingantibody (DART). DARTs are produced as separate polypeptides joined by astabilizing interchain disulphide bond. In exemplary aspects, thebinding agent is a DART comprising an scFv comprising each of the aminoacid sequences of SEQ ID NOs: 1-6 or comprises SEQ ID NO: 7 and/or SEQID NO: 8. Methods of making and using DART antibodies are described inthe art. See, e.g., Rossi et al., MAbs 6: 381-91 (2014); Fournier andSchirrmacher, BioDrugs 27:35-53 (2013); Johnson et al., J Mol Biol399:436-449 (2010); Brien et al., J Virol 87: 7747-7753 (2013); andMoore et al., Blood 117:4542 (2011).

In exemplary aspects, the binding agent is a tetravalent tandem diabody(TandAbs) in which an antibody fragment is produced as a non-covalenthomodimer folder in a head-to-tail arrangement. In exemplary aspects,the binding agent is a TandAbs comprising an scFv comprising each of theamino acid sequences of SEQ ID NOs: 1-6 or comprises SEQ ID NO: 7 and/orSEQ ID NO: 8. TandAbs are known in the art. See, e.g., McAleese et al.,Future Oncol 8: 687-695 (2012); Portner et al., Cancer ImmunolImmunother 61:1869-1875 (2012); and Reusch et al., MAbs 6:728 (2014).

In exemplary aspects, the BiTE, DART, or TandAbs comprises the CDRs ofSEQ ID NOs: 1-6. In exemplary aspects, the BiTE, DART, or TandAbscomprises the amino acid sequence of SEQ ID NOs: 7 and 8. In exemplaryaspects, the BiTE, DART, or TandAbs comprises SEQ ID NOs: 13.

Suitable methods of making antibodies are known in the art. Forinstance, standard hybridoma methods are described in, e.g., Harlow andLane (eds.), Antibodies: A Laboratory Manual, CSH Press (1988), and CA.Janeway et al. (eds.), Immunobiology, 5^(th) Ed., Garland Publishing,New York, N.Y. (2001)).

Monoclonal antibodies for use in the invention may be prepared using anytechnique that provides for the production of antibody molecules bycontinuous cell lines in culture. These include, but are not limited to,the hybridoma technique originally described by Koehler and Milstein(Nature 256: 495-497, 1975), the human B-cell hybridoma technique(Kosbor et al., Immunol Today 4:72, 1983; Cote et al., Proc Natl AcadSci 80: 2026-2030, 1983) and the EBV-hybridoma technique (Cole et al.,Monoclonal Antibodies and Cancer Therapy, Alan R Liss Inc, New YorkN.Y., pp 77-96, (1985).

Briefly, a polyclonal antibody is prepared by immunizing an animal withan immunogen comprising a polypeptide of the present invention andcollecting antisera from that immunized animal. A wide range of animalspecies can be used for the production of antisera. In some aspects, ananimal used for production of anti-antisera is a non-human animalincluding rabbits, mice, rats, hamsters, goat, sheep, pigs or horses.Because of the relatively large blood volume of rabbits, a rabbit, insome exemplary aspects, is a preferred choice for production ofpolyclonal antibodies. In an exemplary method for generating apolyclonal antisera immunoreactive with the chosen IL13Rα2 epitope, 50μg of IL13Rα2 antigen is emulsified in Freund's Complete Adjuvant forimmunization of rabbits. At intervals of, for example, 21 days, 50 μg ofepitope are emulsified in Freund's Incomplete Adjuvant for boosts.Polyclonal antisera may be obtained, after allowing time for antibodygeneration, simply by bleeding the animal and preparing serum samplesfrom the whole blood.

Briefly, in exemplary embodiments, to generate monoclonal antibodies, amouse is injected periodically with recombinant IL13Rα2 against whichthe antibody is to be raised (e.g., 10-20 μg IL13Rα2 emulsified inFreund's Complete Adjuvant). The mouse is given a final pre-fusion boostof a IL13Rα2 polypeptide containing the epitope that allows specificrecognition of lymphatic endothelial cells in PBS, and four days laterthe mouse is sacrificed and its spleen removed. The spleen is placed in10 ml serum-free RPMI 1640, and a single cell suspension is formed bygrinding the spleen between the frosted ends of two glass microscopeslides submerged in serum-free RPMI 1640, supplemented with 2 mML-glutamine, 1 mM sodium pyruvate, 100 units/ml penicillin, and 100μg/ml streptomycin (RPMI) (Gibco, Canada). The cell suspension isfiltered through sterile 70-mesh Nitex cell strainer (Becton Dickinson,Parsippany, N.J.), and is washed twice by centrifuging at 200 g for 5minutes and resuspending the pellet in 20 ml serum-free RPMI.Splenocytes taken from three naive Balb/c mice are prepared in a similarmanner and used as a control. NS-1 myeloma cells, kept in log phase inRPMI with 11% fetal bovine serum (FBS) (Hyclone Laboratories, Inc.,Logan, Utah) for three days prior to fusion, are centrifuged at 200 gfor 5 minutes, and the pellet is washed twice.

Spleen cells (1×10⁸) are combined with 2.0×10⁷ NS-1 cells andcentrifuged, and the supernatant is aspirated. The cell pellet isdislodged by tapping the tube, and 1 ml of 37° C. PEG 1500 (50% in 75 mMHepes, pH 8.0) (Boehringer Mannheim) is added with stirring over thecourse of 1 minute, followed by the addition of 7 ml of serum-free RPMIover 7 minutes. An additional 8 ml RPMI is added and the cells arecentrifuged at 200 g for 10 minutes. After discarding the supernatant,the pellet is resuspended in 200 ml RPMI containing 15% FBS, 100 μMsodium hypoxanthine, 0.4 μM aminopterin, 16 μM thymidine (HAT) (Gibco),25 units/ml IL-6 (Boehringer Mannheim) and 1.5×10⁶ splenocytes/ml andplated into 10 Corning flat-bottom 96-well tissue culture plates(Corning, Corning N.Y.).

On days 2, 4, and 6, after the fusion, 100 μl of medium is removed fromthe wells of the fusion plates and replaced with fresh medium. On day 8,the fusion is screened by ELISA, testing for the presence of mouse IgGbinding to IL13Rα2 as follows. Immulon 4 plates (Dynatech, Cambridge,Mass.) are coated for 2 hours at 37° C. with 100 ng/well of IL13Rα2diluted in 25 mM Tris, pH 7.5. The coating solution is aspirated and 200μl/well of blocking solution (0.5% fish skin gelatin (Sigma) diluted inCMF-PBS) is added and incubated for 30 minutes at 37° C. Plates arewashed three times with PBS containing 0.05% Tween 20 (PBST) and 50 μlculture supernatant is added. After incubation at 37° C. for 30 minutes,and washing as above, 50 μl of horseradish peroxidase-conjugated goatanti-mouse IgG(Fc) (Jackson ImmunoResearch, West Grove, Pa.) diluted1:3500 in PBST is added. Plates are incubated as above, washed fourtimes with PBST, and 100 μl substrate, consisting of 1 mg/ml o-phenylenediamine (Sigma) and 0.1 μl/ml 30% H₂O₂ in 100 mM citrate, pH 4.5, areadded. The color reaction is stopped after 5 minutes with the additionof 50 μl of 15% H₂SO₄. The A₄₉₀ absorbance is determined using a platereader (Dynatech).

Selected fusion wells are cloned twice by dilution into 96-well platesand visual scoring of the number of colonies/well after 5 days. Themonoclonal antibodies produced by hybridomas are isotyped using theIsostrip system (Boehringer Mannheim, Indianapolis, Ind.).

When the hybridoma technique is employed, myeloma cell lines may beused. Such cell lines suited for use in hybridoma-producing fusionprocedures preferably are non-antibody-producing, have high fusionefficiency, and enzyme deficiencies that render them incapable ofgrowing in certain selective media that support the growth of only thedesired fused cells (hybridomas). For example, where the immunizedanimal is a mouse, one may use P3-X63/Ag8, P3-X63-Ag8.653, NS1/1.Ag 4 1,Sp210-Ag14, FO, NSO/U, MPC-11, MPC11-X45-GTG 1.7 and S194/15XX0 Bul; forrats, one may use R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210; and U-266,GM1500-GRG2, LICR-LON-HMy2 and UC729-6 are all useful in connection withcell fusions. It should be noted that the hybridomas and cell linesproduced by such techniques for producing the monoclonal antibodies arecontemplated to be compositions of the disclosure.

Depending on the host species, various adjuvants may be used to increasean immunological response. Such adjuvants include, but are not limitedto, Freund's, mineral gels such as aluminum hydroxide, and surfaceactive substances such as lysolecithin, pluronic polyols, polyanions,peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol.BCG (bacilli Calmette-Guerin) and Corynebacterium parvum are potentiallyuseful human adjuvants.

Alternatively, other methods, such as EBV-hybridoma methods (Haskard andArcher, J. Immunol. Methods, 74(2), 361-67 (1984),and Roder et al.₅Methods Enzymol., 121, 140-67 (1986)), and bacteriophage vectorexpression systems (see, e.g., Huse et al., Science, 246, 1275-81(1989)) that are known in the art may be used. Further, methods ofproducing antibodies in non-human animals are described in, e.g., U.S.Pat. Nos. 5,545,806, 5,569,825, and 5,714,352, and U.S. PatentApplication Publication No. 2002/0197266 A1).

Antibodies may also be produced by inducing in vivo production in thelymphocyte population or by screening recombinant immunoglobulinlibraries or panels of highly specific binding reagents as disclosed inOrlandi et al. (Proc. Natl. Acad. Sci. 86: 3833-3837; 1989), and Winterand Milstein (Nature 349: 293-299, 1991).

Furthermore, phage display can be used to generate an antibody of thedisclosure. In this regard, phage libraries encoding antigen-bindingvariable (V) domains of antibodies can be generated using standardmolecular biology and recombinant DNA techniques (see, e.g., Sambrook etal. (eds.), Molecular Cloning, A Laboratory Manual, 3^(rd) Edition, ColdSpring Harbor Laboratory Press, New York (2001)). Phage encoding avariable region with the desired specificity are selected for specificbinding to the desired antigen, and a complete or partial antibody isreconstituted comprising the selected variable domain. Nucleic acidsequences encoding the reconstituted antibody are introduced into asuitable cell line, such as a myeloma cell used for hybridomaproduction, such that antibodies having the characteristics ofmonoclonal antibodies are secreted by the cell (see, e.g., Janeway etal., supra, Huse et al., supra, and U.S. Pat. No. 6,265,150). Relatedmethods also are described in U.S. Pat. Nos. 5,403,484; 5,571,698;5,837,500; and 5,702,892. The techniques described in U.S. Pat. Nos.5,780,279; 5,821,047; 5,824,520; 5,855,885; 5,858,657; 5,871,907;5,969,108; 6,057,098; and 6,225,447, are also contemplated as useful inpreparing antibodies according to the disclosure.

Antibodies can be produced by transgenic mice that are transgenic forspecific heavy and light chain immunoglobulin genes. Such methods areknown in the art and described in, for example U.S. Pat. Nos. 5,545,806and 5,569,825, and Janeway et al., supra.

Methods for generating humanized antibodies are well known in the artand are described in detail in, for example, Janeway et al., supra, U.S.Pat. Nos. 5,225,539; 5,585,089; and 5,693,761; European Patent No.0239400 B1; and United Kingdom Patent No. 2188638. Humanized antibodiescan also be generated using the antibody resurfacing technologydescribed in U.S. Pat. No. 5,639,641 and Pedersen et al., J. Mol. Biol.,235:959-973 (1994).

Techniques developed for the production of “chimeric antibodies,” thesplicing of mouse antibody genes to human antibody genes to obtain amolecule with appropriate antigen specificity and biological activity,can be used (Morrison et al., Proc. Natl. Acad. Sci. 81: 6851-6855,1984; Neuberger et al., Nature 312: 604-608, 1984; and Takeda et al.,Nature 314: 452-454; 1985). Alternatively, techniques described for theproduction of single-chain antibodies (U.S. Pat. No. 4,946,778) can beadapted to produce IL13Rα2-specific single chain antibodies.

A preferred chimeric or humanized antibody has a human constant region,while the variable region, or at least a CDR, of the antibody is derivedfrom a non-human species. Methods for humanizing non-human antibodiesare well known in the art. (see U.S. Pat. Nos. 5,585,089, and5,693,762). Generally, a humanized antibody has one or more amino acidresidues introduced into a CDR region and/or into its framework regionfrom a source which is non-human. Humanization can be performed, forexample, using methods described in Jones et al. (Nature 321: 522-525,1986), Riechmann et al., (Nature, 332: 323-327, 1988) and Verhoeyen etal. (Science 239:1534-1536, 1988), by substituting at least a portion ofa rodent complementarity-determining region (CDR) for the correspondingregion of a human antibody. Numerous techniques for preparing engineeredantibodies are described, e.g., in Owens and Young, J. Immunol. Meth.,168:149-165 (1994). Further changes can then be introduced into theantibody framework to modulate affinity or immunogenicity.

Consistent with the foregoing description, compositions comprising CDRsmay be generated using, at least in part, techniques known in the art toisolate CDRs. Complementarity-determining regions are characterized bysix polypeptide loops, three loops for each of the heavy or light chainvariable regions. The amino acid position in a CDR is defined by Kabatet al., “Sequences of Proteins of Immunological Interest,” U.S.Department of Health and Human Services, (1983), which is incorporatedherein by reference. For example, hypervariable regions of humanantibodies are roughly defined to be found at residues 28 to 35, from49-59 and from residues 92-103 of the heavy and light chain variableregions [Janeway et al., supra]. The murine CDRs also are found atapproximately these amino acid residues. It is understood in the artthat CDR regions may be found within several amino acids of theapproximated amino acid positions set forth above. An immunoglobulinvariable region also consists of four “framework” regions surroundingthe CDRs (FR1-4). The sequences of the framework regions of differentlight or heavy chains are highly conserved within a species, and arealso conserved between human and murine sequences.

Compositions comprising one, two, and/or three CDRs of a heavy chainvariable region or a light chain variable region of a monoclonalantibody are generated. For example, using antibody of hybridoma clone47 comprising the CDRs having the sequences of SEQ ID NOs: 1-6,polypeptide compositions comprising these CDRs are generated.Polypeptide compositions comprising one, two, three, four, five and/orsix complementarity-determining regions of an antibody are alsocontemplated. Using the conserved framework sequences surrounding theCDRs, PCR primers complementary to these consensus framework sequencesare generated to amplify the CDR sequence located between the primerregions. Techniques for cloning and expressing nucleotide andpolypeptide sequences are well-established in the art [see e.g.,Sambrook et al., Molecular Cloning: A Laboratory Manual, 2^(nd) Edition,Cold Spring Harbor, N.Y. (1989)]. The amplified CDR sequences areligated into an appropriate plasmid. The plasmid comprising one, two,three, four, five and/or six cloned CDRs optionally contains additionalpolypeptide encoding regions linked to the CDR.

It is contemplated that modified polypeptide compositions comprisingone, two, three, four, five, or six CDRs of a heavy or light chain ofSEQ ID NOs: 1-6 are generated, wherein a CDR is altered to provideincreased specificity or affinity or avidity to the target IL13Rα2.Sites at locations in the CDRs are typically modified in series, e.g.,by substituting first with conservative choices (e.g., hydrophobic aminoacid substituted for a non-identical hydrophobic amino acid) and thenwith more dissimilar choices (e.g., hydrophobic amino acid substitutedfor a charged amino acid), and then deletions or insertions may be madeat the target site.

Framework regions (FR) of a murine antibody are humanized bysubstituting compatible human framework regions chosen from a largedatabase of human antibody variable sequences, including over twelvehundred human V_(H) sequences and over one thousand V_(L) sequences. Thedatabase of antibody sequences used for comparison is downloaded fromAndrew C. R. Martin's KabatMan web page(http://www.rubic.rdg.ac.uk/abs/). The Kabat method for identifying CDRsprovides a means for delineating the approximate CDR and frameworkregions of any human antibody and comparing the sequence of a murineantibody for similarity to determine the CDRs and FRs. Best matchedhuman V_(H) and V_(L) sequences are chosen on the basis of high overallframework matching, similar CDR length, and minimal mismatching ofcanonical and V_(H)/V_(L) contact residues. Human framework regions mostsimilar to the murine sequence are inserted between the murine CDRs.Alternatively, the murine framework region may be modified by makingamino acid substitutions of all or part of the native framework regionthat more closely resemble a framework region of a human antibody.

“Conservative” amino acid substitutions are made on the basis ofsimilarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues involved.For example, nonpolar (hydrophobic) amino acids include alanine (Ala,A), leucine (Leu, L), isoleucine (Ile, I), valine (Val, V), proline(Pro, P), phenylalanine (Phe, F), tryptophan (Trp, W), and methionine(Met, M); polar neutral amino acids include glycine (Gly, G), serine(Ser, S), threonine (Thr, T), cysteine (Cys, C), tyrosine (Tyr, Y),asparagine (Asn, N), and glutamine (Gln, Q); positively charged (basic)amino acids include arginine (Arg, R), lysine (Lys, K), and histidine(His, H); and negatively charged (acidic) amino acids include asparticacid (Asp, D) and glutamic acid (Glu, E). “Insertions” or “deletions”are preferably in the range of about 1 to 20 amino acids, morepreferably 1 to 10 amino acids. The variation may be introduced bysystematically making substitutions of amino acids in a polypeptidemolecule using recombinant DNA techniques and assaying the resultingrecombinant variants for activity. Nucleic acid alterations can be madeat sites that differ in the nucleic acids from different species(variable positions) or in highly conserved regions (constant regions).Methods for expressing polypeptide compositions useful in the inventionare described in greater detail below.

Additionally, another useful technique for generating antibodies for usein the methods of the disclosure may be one which uses a rationaldesign-type approach. The goal of rational design is to producestructural analogs of biologically active polypeptides or compounds withwhich they interact (agonists, antagonists, inhibitors, peptidomimetics,binding partners, and the like). In this case, the active polypeptidescomprise the sequences of SEQ ID NOs: 1-6 disclosed herein. By creatingsuch analogs, it is possible to fashion additional antibodies which aremore immunoreactive than the native or natural molecule. In oneapproach, one would generate a three-dimensional structure for theantibodies or an epitope binding fragment thereof. This could beaccomplished by x-ray crystallography, computer modeling or by acombination of both approaches. An alternative approach, “alanine scan,”involves the random replacement of residues throughout a molecule withalanine, and the resulting effect on function is determined.

It also is possible to solve the crystal structure of the specificantibodies. In principle, this approach yields a pharmacore upon whichsubsequent drug design can be based. It is possible to bypass proteincrystallography altogether by generating anti-idiotypic antibodies to afunctional, pharmacologically active antibody. As a mirror image of amirror image, the binding site of anti-idiotype antibody is expected tobe an analog of the original antigen. The anti-idiotype antibody is thenbe used to identify and isolate additional antibodies from banks ofchemically- or biologically-produced peptides.

Chemically synthesized bispecific antibodies may be prepared bychemically crosslinking heterologous Fab or F(ab′)₂ fragments by meansof chemicals such as heterobifunctional reagentsuccinimidyl-3-(2-pyridyldithiol)-propionate (SPDP, Pierce Chemicals,Rockford, Ill.). The Fab and F(ab′)₂ fragments can be obtained fromintact antibody by digesting it with papain or pepsin, respectively(Karpovsky et al., J. Exp. Med. 160:1686-701, 1984; Titus et al., J.Immunol., 138:4018-22, 1987).

Methods of testing antibodies for the ability to bind to the epitope ofthe IL13Rα2, regardless of how the antibodies are produced, are known inthe art and include any antibody-antigen binding assay such as, forexample, radioimmunoassay (MA), ELISA, Western blot,immunoprecipitation, and competitive inhibition assays (see, e.g.,Janeway et al., infra, and U.S. Patent Application Publication No.2002/0197266 A1).

Selection of antibodies from an antibody population for purposes hereinalso include using blood vessel endothelial cells to “subtract” thoseantibodies that cross-react with epitopes on such cells other thanIL13Rα2 epitopes. The remaining antibody population is enriched inantibodies preferential for IL13Rα2 epitopes.

Aptamers

Recent advances in the field of combinatorial sciences have identifiedshort polymer sequences (e.g., oligonucleic acid or peptide molecules)with high affinity and specificity to a given target. For example, SELEXtechnology has been used to identify DNA and RNA aptamers with bindingproperties that rival mammalian antibodies, the field of immunology hasgenerated and isolated antibodies or antibody fragments which bind to amyriad of compounds, and phage display has been utilized to discover newpeptide sequences with very favorable binding properties. Based on thesuccess of these molecular evolution techniques, it is certain thatmolecules can be created which bind to any target molecule. A loopstructure is often involved with providing the desired bindingattributes as in the case of aptamers, which often utilize hairpin loopscreated from short regions without complementary base pairing, naturallyderived antibodies that utilize combinatorial arrangement of loopedhyper-variable regions and new phage-display libraries utilizing cyclicpeptides that have shown improved results when compared to linearpeptide phage display results. Thus, sufficient evidence has beengenerated to indicate that high affinity ligands can be created andidentified by combinatorial molecular evolution techniques. For thepresent disclosure, molecular evolution techniques can be used toisolate binding agents specific for the IL13Rα2 disclosed herein. Formore on aptamers, see generally, Gold, L., Singer, B., He, Y. Y., Brody.E., “Aptamers As Therapeutic And Diagnostic Agents,” J. Biotechnol.74:5-13 (2000). Relevant techniques for generating aptamers are found inU.S. Pat. No. 6,699,843, which is incorporated herein by reference inits entirety.

In some embodiments, the aptamer is generated by preparing a library ofnucleic acids; contacting the library of nucleic acids with a growthfactor, wherein nucleic acids having greater binding affinity for thegrowth factor (relative to other library nucleic acids) are selected andamplified to yield a mixture of nucleic acids enriched for nucleic acidswith relatively higher affinity and specificity for binding to thegrowth factor. The processes may be repeated, and the selected nucleicacids mutated and rescreened, whereby a growth factor aptamer isidentified. Nucleic acids may be screened to select for molecules thatbind to more than target. Binding more than one target can refer tobinding more than one simultaneously or competitively. In someembodiments, a binding agent comprises at least one aptamer, wherein afirst binding unit binds a first epitope of an IL13Rα2 and a secondbinding unit binds a second epitope of the IL13Rα2.

With regard to the binding agents of the compositions of the disclosure,ligand-induced activation of the IL13Rα2 is reduced upon binding of thebinding agent to the IL13Rα2. As used herein, the term “reduce” as wellas like terms, e.g., “inhibit,” do not necessarily imply 100% or acomplete reduction or inhibition. Rather, there are varying degrees ofreduction or inhibition of which one of ordinary skill in the artrecognizes as having a potential benefit or therapeutic effect.Accordingly, in some embodiments, ligand-induced activation of theIL13Rα2 is completely abolished. In some embodiments, ligand-inducedactivation is substantially reduced, e.g., reduced by about 10% (e.g.,by about 20%, by about 30%, by about 40%, by about 50%, by about 60%, byabout 70%, by about 80%, by about 90%) or more, as compared toligand-induced activation of the IL13Rα2 when the binding agent isabsent or not bound to the IL13Rα2. Methods of measuring ligand-inducedactivation of an IL13Rα2 are known in the art, and include, for example,the assays described in the Examples, below.

Conjugates

Conjugates comprising a targeting domain and an effector domain aredisclosed herein. In exemplary embodiments, the conjugate comprises anyone of the binding agents disclosed herein as the targeting domain tolocalize the conjugate to a cell expressing IL13Rα2, e.g., a tumor cellexpressing the same, and an effector domain. In exemplary aspects, theconjugate is a fusion protein. In exemplary aspects, the conjugate is achimeric protein. As used herein, the term “chimeric” refers to amolecule composed of parts of different origins. A chimeric molecule, asa whole, is non-naturally occurring, e.g., synthetic or recombinant,although the parts which comprise the chimeric molecule may be naturallyoccurring.

Exemplary Effector Domains

As used herein, the term “effector domain” refers to a portion of aconjugate that effects a desired biological function. In exemplaryaspects, the effector domain identifies or locates IL13Rα2-expressingcells. For example, the effector domain may be a diagnostic agent, e.g.,a radiolabel, a fluorescent label, an enzyme (e.g., that catalyzes acalorimetric or fluorometric reaction), a substrate, a solid matrix, ora carrier (e.g., biotin or avidin). The diagnostic agent in some aspectsis an imaging agent. Many appropriate imaging agents are known in theart, as are methods of attaching the labeling agents to the peptides ofthe invention (see, e.g., U.S. Pat. Nos. 4,965,392; 4,472,509;5,021,236; and 5,037,630; each incorporated herein by reference). Theimaging agents are administered to a subject in a pharmaceuticallyacceptable carrier, and allowed to accumulate at a target site havingthe lymphatic endothelial cells. This imaging agent then serves as acontrast reagent for X-ray, magnetic resonance, positron emissiontomography, single photon emission computed tomography (SPECT), orsonographic or scintigraphic imaging of the target site. Of course, itshould be understood that the imaging may be performed in vitro wheretissue from the subject is obtained through a biopsy, and the presenceof lymphatic endothelial cells is determined with the aid of the imagingagents described herein in combination with histochemical techniques forpreparing and fixing tissues. Paramagnetic ions useful in the imagingagents of the invention include for example chromium (III), manganese(II), iron (III), iron (II), cobalt (II), nickel (II) copper (II),neodymium (III), samarium (III), ytterbium(III), gadolinium (III),vanadium (II), terbium (III), dysprosium (III), holmium (III) and erbium(III). Ions useful for X-ray imaging include, but are not limited to,lanthanum (III), gold (III), lead (II) and particularly bismuth (III).Radioisotopes for diagnostic applications include for example,²¹¹astatine, ¹⁴carbon, ⁵¹chromium, ³⁶chlorine, ⁵⁷cobalt, ⁶⁷copper,¹⁵²europium, ⁶⁷gallium, ³hydrogen, ¹²³iodine, ¹²⁵iodine, ¹¹¹indium,⁵⁹iron, ³²phosphorus, ¹⁸⁶rhenium, ⁷⁵selenium, ³⁵sulphur, ⁹⁹mtechnicium,⁹⁰yttrium, and ⁹⁹zirconium.

The effector domain may be one which alters the physico-chemicalcharacteristics of the conjugate, e.g., an effector which confersincreased solubility and/or stability and/or half-life, resistance toproteolytic cleavage, modulation of clearance. In exemplary aspects, theeffector domain is a polymer, a carbohydrate, or a lipid.

The polymer may be branched or unbranched. The polymer may be of anymolecular weight. The polymer in some embodiments has an averagemolecular weight of between about 2 kDa to about 100 kDa (the term“about” indicating that in preparations of a water-soluble polymer, somemolecules will weigh more, some less, than the stated molecular weight).The average molecular weight of the polymer is in some aspects betweenabout 5 kDa and about 50 kDa, between about 12 kDa to about 40 kDa orbetween about 20 kDa to about 35 kDa. In some embodiments, the polymeris modified to have a single reactive group, such as an active ester foracylation or an aldehyde for alkylation, so that the degree ofpolymerization may be controlled. The polymer in some embodiments iswater soluble so that the protein to which it is attached does notprecipitate in an aqueous environment, such as a physiologicalenvironment. In some embodiments when, for example, the composition isused for therapeutic use, the polymer is pharmaceutically acceptable.Additionally, in some aspects, the polymer is a mixture of polymers,e.g., a co-polymer, a block co-polymer. In some embodiments, the polymeris selected from the group consisting of: polyamides, polycarbonates,polyalkylenes and derivatives thereof, including polyalkylene glycols,polyalkylene oxides, polyalkylene terepthalates, polymers of acrylic andmethacrylic esters, including poly(methyl methacrylate), poly(ethylmethacrylate), poly(butylmethacrylate), poly(isobutyl methacrylate),poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(laurylmethacrylate), poly(phenyl methacrylate), poly(methyl acrylate),poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecylacrylate), polyvinyl polymers including polyvinyl alcohols, polyvinylethers, polyvinyl esters, polyvinyl halides, poly(vinyl acetate), andpolyvinylpyrrolidone, polyglycolides, polysiloxanes, polyurethanes andco-polymers thereof, celluloses including alkyl cellulose, hydroxyalkylcelluloses, cellulose ethers, cellulose esters, nitro celluloses, methylcellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxy-propylmethyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate,cellulose propionate, cellulose acetate butyrate, cellulose acetatephthalate, carboxylethyl cellulose, cellulose triacetate, and cellulosesulphate sodium salt, polypropylene, polyethylenes includingpoly(ethylene glycol), poly(ethylene oxide), and poly(ethyleneterephthalate), and polystyrene. In some aspects, the polymer is abiodegradable polymer, including a synthetic biodegradable polymer(e.g., polymers of lactic acid and glycolic acid, polyanhydrides,poly(ortho)esters, polyurethanes, poly(butic acid), poly(valeric acid),and poly(lactide-cocaprolactone)), and a natural biodegradable polymer(e.g., alginate and other polysaccharides including dextran andcellulose, collagen, chemical derivatives thereof (substitutions,additions of chemical groups, for example, alkyl, alkylene,hydroxylations, oxidations, and other modifications routinely made bythose skilled in the art), albumin and other hydrophilic proteins (e.g.,zein and other prolamines and hydrophobic proteins)), as well as anycopolymer or mixture thereof. In general, these materials degrade eitherby enzymatic hydrolysis or exposure to water in vivo, by surface or bulkerosion. In some aspects, the polymer is a bioadhesive polymer, such asa bioerodible hydrogel described by H. S. Sawhney, C. P. Pathak and J.A. Hubbell in Macromolecules, 1993, 26, 581-587, the teachings of whichare incorporated herein, polyhyaluronic acids, casein, gelatin, glutin,polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methylmethacrylates), poly(ethyl methacrylates), poly(butylmethacrylate),poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecylmethacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate),poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutylacrylate), and poly(octadecyl acrylate). In some embodiments, thepolymer is a water-soluble polymer or a hydrophilic polymer. Suitablewater-soluble polymers are known in the art and include, for example,polyvinylpyrrolidone, hydroxypropyl cellulose (HPC; Klucel),hydroxypropyl methylcellulose (HPMC; Methocel), nitrocellulose,hydroxypropyl ethylcellulose, hydroxypropyl butylcellulose,hydroxypropyl pentylcellulose, methyl cellulose, ethylcellulose(Ethocel), hydroxyethyl cellulose, various alkyl celluloses andhydroxyalkyl celluloses, various cellulose ethers, cellulose acetate,carboxymethyl cellulose, sodium carboxymethyl cellulose, calciumcarboxymethyl cellulose, vinyl acetate/crotonic acid copolymers,poly-hydroxyalkyl methacrylate, hydroxymethyl methacrylate, methacrylicacid copolymers, polymethacrylic acid, polymethylmethacrylate, maleicanhydride/methyl vinyl ether copolymers, poly vinyl alcohol, sodium andcalcium polyacrylic acid, polyacrylic acid, acidic carboxy polymers,carboxypolymethylene, carboxyvinyl polymers, polyoxyethylenepolyoxypropylene copolymer, polymethylvinylether co-maleic anhydride,carboxymethylamide, potassium methacrylate divinylbenzene co-polymer,polyoxyethyleneglycols, polyethylene oxide, and derivatives, salts, andcombinations thereof. In some aspects, the water-soluble polymers ormixtures thereof include, but are not limited to, N-linked or O-linkedcarbohydrates, sugars, phosphates, carbohydrates; sugars; phosphates;polyethylene glycol (PEG) (including the forms of PEG that have beenused to derivatize proteins, including mono-(C1-C 10) alkoxy- oraryloxy-polyethylene glycol); monomethoxy-polyethylene glycol; dextran(such as low molecular weight dextran of, for example, about 6 kD),cellulose; other carbohydrate-based polymers, poly-(N-vinylpyrrolidone), polyethylene glycol, propylene glycol homopolymers, apolypropylene oxide/ethylene oxide co-polymer, polyoxyethylated polyols(e.g., glycerol) and polyvinyl alcohol. Also encompassed by thedisclosure are bifunctional crosslinking molecules which may be used toprepare covalently attached multimers. A particularly preferredwater-soluble polymer for use herein is polyethylene glycol (PEG). Asused herein, polyethylene glycol is meant to encompass any of the formsof PEG that can be used to derivatize other proteins, such asmono-(C1-C10) alkoxy- or aryloxy-polyethylene glycol. PEG is a linear orbranched neutral polyether, available in a broad range of molecularweights, and is soluble in water and most organic solvents. PEG iseffective at excluding other polymers or peptides when present in water,primarily through its high dynamic chain mobility and hydrophobicnature, thus creating a water shell or hydration sphere when attached toother proteins or polymer surfaces. PEG is nontoxic, non-immunogenic,and approved by the Food and Drug Administration for internalconsumption. Proteins or enzymes when conjugated to PEG havedemonstrated bioactivity, non-antigenic properties, and decreasedclearance rates when administered in animals. F. M. Veronese et al.,Preparation and Properties of Monomethoxypoly(ethylene glycol)-modifiedEnzymes for Therapeutic Applications, in J. M. Harris ed., Poly(EthyleneGlycol) Chemistry—Biotechnical and Biomedical Applications, 127-36,1992, incorporated herein by reference. Without wishing to be bound bytheory, these phenomena may be due to the exclusion properties of PEG inpreventing recognition by the immune system. In addition, PEG has beenwidely used in surface modification procedures to decrease proteinadsorption and improve blood compatibility. S. W. Kim et al., Ann. N.Y.Acad. Sci. 516: 116-30 1987; Jacobs et al., Artif. Organs 12: 500-501,1988; Park et al., J. Poly. Sci, Part A 29:1725-31, 1991, eachincorporated herein by reference in its entirety. Hydrophobic polymersurfaces, such as polyurethanes and polystyrene, can be modified by thegrafting of PEG (MW 3,400) and employed as nonthrombogenic surfaces.Surface properties (contact angle) can be more consistent withhydrophilic surfaces, due to the hydrating effect of PEG. Moreimportantly, protein (albumin and other plasma proteins) adsorption canbe greatly reduced, resulting from the high chain motility, hydrationsphere, and protein exclusion properties of PEG. PEG (MW 3,400) wasdetermined as an optimal size in surface immobilization studies, Park etal., J. Biomed. Mat. Res. 26:739-45, 1992, while PEG (MW 5,000) was mostbeneficial in decreasing protein antigenicity. F. M. Veronese et al., InJ. M. Harris, et al., Poly(Ethylene Glycol) Chemistry—Biotechnical andBiomedical Applications, 127-36. Methods for preparing pegylated bindingagent polypeptides may comprise the steps of (a) reacting thepolypeptide with polyethylene glycol (such as a reactive ester oraldehyde derivative of PEG) under conditions whereby the binding agentpolypeptide becomes attached to one or more PEG groups, and (b)obtaining the reaction product(s). In general, the optimal reactionconditions for the acylation reactions will be determined based on knownparameters and the desired result. For example, the larger the ratio ofPEG:protein, the greater the percentage of poly-pegylated product. Insome embodiments, the binding agent will have a single PEG moiety at theN-terminus. See U.S. Pat. No. 8,234,784, incorporated by referenceherein.

In some embodiments, the effector domain is a carbohydrate. In someembodiments, the carbohydrate is a monosaccharide (e.g., glucose,galactose, fructose), a disaccharide (e.g., sucrose, lactose, maltose),an oligosaccharide (e.g., raffinose, stachyose), a polysaccharide (e.g.,a starch, amylase, amylopectin, cellulose, chitin, callose, laminarin,xylan, mannan, fucoidan, or galactomannan).

In some embodiments, the effector domain is a lipid. The lipid, in someembodiments, is a fatty acid, eicosanoid, prostaglandin, leukotriene,thromboxane, N-acyl ethanolamine, glycerolipid (e.g., mono-, di-,tri-substituted glycerols), glycerophospholipid (e.g.,phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine,phosphatidylserine), sphingolipid (e.g., sphingosine, ceramide), sterollipid (e.g., steroid, cholesterol), prenol lipid, saccharolipid, or apolyketide, oil, wax, cholesterol, sterol, fat-soluble vitamin,monoglyceride, diglyceride, triglyceride, or a phospholipid.

Lethal Domains

In exemplary aspects, the effector domain is a lethal domain thatconfers lethality, such that when the conjugate is localized to a cellexpressing IL13Rα2, e.g., a tumor cell expressing the same. The effectordomain confers upon the conjugate the ability to kill anIL13Rα2-expressing cell once the binding agent has found and bound toits IL13Rα2 target.

In exemplary aspects, the effector domain is a cytotoxin (also referredto herein as a “cytotoxic agent”). The cytotoxic agent is any molecule(chemical or biochemical) which is toxic to a cell. In some embodiments,the cytotoxic agent is a chemotherapeutic agent. Chemotherapeutic agentsare known in the art and include, but are not limited to, platinumcoordination compounds, topoisomerase inhibitors, antibiotics,antimitotic alkaloids and difluoronucleosides, as described in U.S. Pat.No. 6,630,124. In some embodiments, the chemotherapeutic agent is aplatinum coordination compound. The term “platinum coordinationcompound” refers to any tumor cell growth-inhibiting platinumcoordination compound that provides the platinum in the form of an ion.In some embodiments, the platinum coordination compound iscis-diamminediaquoplatinum (II)-ion;chloro(diethylenetriamine)-platinum(II)chloride;dichloro(ethylenediamine)-platinum(II),diammine(1,1-cyclobutanedicarboxylato) platinum(II) (carboplatin);spiroplatin; iproplatin; diammine(2-ethylmalonato)-platinum(II);ethylenediaminemalonatoplatinum(II);aqua(1,2-diaminodyclohexane)-sulfatoplatinum(II);(1,2-diaminocyclohexane)malonatoplatinum(II);(4-caroxyphthalato)(1,2-diaminocyclohexane)platinum(II);(1,2-diaminocyclohexane)-(isocitrato)platinum(II);(1,2-diaminocyclohexane)cis(pyruvato)platinum(II);(1,2-diaminocyclohexane)oxalatoplatinum(II); ormaplatin; or tetraplatin.In some embodiments, cisplatin is the platinum coordination compoundemployed in the compositions and methods of the present invention.Cisplatin is commercially available under the name PLATINOL™ fromBristol Myers-Squibb Corporation and is available as a powder forconstitution with water, sterile saline or other suitable vehicle. Otherplatinum coordination compounds suitable for use in the presentinvention are known and are available commercially and/or can beprepared by conventional techniques. Cisplatin, orcis-dichlorodiammineplatinum II, has been used successfully for manyyears as a chemotherapeutic agent in the treatment of various humansolid malignant tumors. More recently, other diamino-platinum complexeshave also shown efficacy as chemotherapeutic agents in the treatment ofvarious human, solid, malignant tumors. Such diamino-platinum complexesinclude, but are not limited to, spiroplatinum and carboplatinum.Although cisplatin and other diamino-platinum complexes have been widelyused as chemotherapeutic agents in humans, they have had to be deliveredat high dosage levels that can lead to toxicity problems such as kidneydamage.

In some embodiments, the chemotherapeutic agent is a topoisomeraseinhibitor. Topoisomerases are enzymes that are capable of altering DNAtopology in eukaryotic cells. They are critical for cellular functionsand cell proliferation. Generally, there are two classes oftopoisomerases in eukaryotic cells, type I and type II. Topoisomerase Iis a monomeric enzyme of approximately 100,000 molecular weight. Theenzyme binds to DNA and introduces a transient single-strand break,unwinds the double helix (or allows it to unwind), and subsequentlyreseals the break before dissociating from the DNA strand. Varioustopoisomerase inhibitors have recently shown clinical efficacy in thetreatment of humans afflicted with ovarian cancer, esophageal cancer ornon-small cell lung carcinoma. In some aspects, the topoisomeraseinhibitor is camptothecin or a camptothecin analog. Camptothecin is awater-insoluble, cytotoxic alkaloid produced by Camptotheca accuminatatrees indigenous to China and Nothapodytes foetida trees indigenous toIndia. Camptothecin exhibits tumor cell growth-inhibiting activityagainst a number of tumor cells. Compounds of the camptothecin analogclass are typically specific inhibitors of DNA topoisomerase I. By theterm “inhibitor of topoisomerase” is meant any tumor cellgrowth-inhibiting compound that is structurally related to camptothecin.Compounds of the camptothecin analog class include, but are not limitedto; topotecan, irinotecan and 9-amino-camptothecin. In additionalembodiments, the cytotoxic agent is any tumor cell growth-inhibitingcamptothecin analog claimed or described in U.S. Pat. No. 5,004,758;European Patent Application Number 88311366.4 (Publication Number EP 0321 122); U.S. Pat. No. 4,604,463; European Patent ApplicationPublication Number EP 0 137 145; U.S. Pat. No. 4,473,692; EuropeanPatent Application Publication Number EP 0 074 256; U.S. Pat. No.4,545,880; European Patent Application Publication Number EP 0 074 256;European Patent Application Publication Number EP 0 088 642; Wani etal., J. Med. Chem., 29, 2358-2363 (1986); and Nitta et al., Proc. 14thInternational Congr. Chemotherapy, Kyoto, 1985, Tokyo Press, AnticancerSection 1, p. 28-30. In particular, the disclosure contemplates acompound called CPT-11. CPT-11 is a camptothecin analog with a4-(piperidino)-piperidine side chain joined through a carbamate linkageat C-10 of 10-hydroxy-7-ethyl camptothecin. CPT-11 is currentlyundergoing human clinical trials and is also referred to as irinotecan;Wani et al, J. Med. Chem., 23, 554 (1980); Wani et al., J. Med. Chem.,30, 1774 (1987); U.S. Pat. No. 4,342,776; European Patent ApplicationPublication Number EP 418 099; U.S. Pat. No. 4,513,138; European PatentApplication Publication Number EP 0 074 770; U.S. Pat. No. 4,399,276;European Patent Application Publication Number 0 056 692; the entiredisclosure of each of which is hereby incorporated by reference. All ofthe above-listed compounds of the camptothecin analog class areavailable commercially and/or can be prepared by conventional techniquesincluding those described in the above-listed references. Thetopoisomerase inhibitor may be selected from the group consisting oftopotecan, irinotecan and 9-aminocamptothecin.

The preparation of numerous compounds of the camptothecin analog class(including pharmaceutically acceptable salts, hydrates and solvatesthereof) as well as the preparation of oral and parenteralpharmaceutical compositions comprising such a compound of thecamptothecin analog class and an inert, pharmaceutically acceptablecarrier or diluent, is extensively described in U.S. Pat. No. 5,004,758;and European Patent Application Number 88311366.4(Publication Number EP0 321 122), the teachings of each of which are incorporated herein byreference in its entirety.

In still another embodiment of the invention, the chemotherapeutic agentis an antibiotic compound. Suitable antibiotics include, but are notlimited to, doxorubicin, mitomycin, bleomycin, daunorubicin andstreptozocin. In some embodiments, the chemotherapeutic agent is anantimitotic alkaloid. In general, antimitotic alkaloids can be extractedfrom Cantharanthus roseus, and have been shown to be efficacious asanticancer chemotherapy agents. A great number of semi-syntheticderivatives have been studied both chemically and pharmacologically(see, O. Van Tellingen et al, Anticancer Research, 12, 1699-1716(1992)). The antimitotic alkaloids of the present invention include, butare not limited to, vinblastine, vincristine, vindesine, Taxol andvinorelbine. The latter two antimitotic alkaloids are commerciallyavailable from Eli Lilly and Company, and Pierre Fabre Laboratories,respectively (see, U.S. Pat. No. 5,620,985). In one aspect of thedisclosure, the antimitotic alkaloid is vinorelbine.

In another embodiment of the invention, the chemotherapeutic agent is adifluoronucleoside. 2′-deoxy-2′,2′-difluoronucleosides are known in theart as having antiviral activity. Such compounds are disclosed andtaught in U.S. Pat. Nos. 4,526,988 and 4,808,614. European PatentApplication Publication 184,365 discloses that these samedifluoronucleosides have oncolytic activity. In certain specificaspects, the 2′-deoxy-2′,2′-difluoronucleoside used in the compositionsand methods of the disclosure is 2′-deoxy-2′,2′-difluorocytidinehydrochloride, also known as gemcitabine hydrochloride. Gemcitabine iscommercially available or can be synthesized in a multi-step process asdisclosed in U.S. Pat. Nos. 4,526,988, 4,808,614 and 5,223,608, theteachings of each of which are incorporated herein by reference in itsentirety.

In exemplary aspects, the effector domain is an apoptosis tag whichcauses the IL13Rα2-expressing cell to apoptose. In exemplary aspects,the apoptosis tag is a TRAIL protein, or a portion thereof. In exemplaryaspects, the apoptosis tag comprises the amino acid sequence of SEQ IDNO: 27. In exemplary aspects, the conjugate comprises the amino acidsequence of SEQ ID NO: 25.

In exemplary embodiments, the effector domain is an Fc domain of IgG orother immunoglobulin. For substituents such as an Fc region of humanIgG, the fusion can be fused directly to a binding agent or fusedthrough an intervening sequence. For example, a human IgG hinge, CH2 andCH3 region may be fused at either the N-terminus or C-terminus of abinding agent to attach the Fc region. The resulting Fc-fusion agentenables purification via a Protein A affinity column (Pierce, Rockford,Ill.). Peptide and proteins fused to an Fc region can exhibit asubstantially greater half-life in vivo than the unfused counterpart. Afusion to an Fc region allows for dimerization/multimerization of thefusion polypeptide. The Fc region may be a naturally occurring Fcregion, or may be modified for superior characteristics, e.g.,therapeutic qualities, circulation time, reduced aggregation. As notedabove, in some embodiments, the binding agent are conjugated, e.g.,fused to an immunoglobulin or portion thereof (e.g., variable region,CDR, or Fc region). Known types of immunoglobulins (Ig) include IgG,IgA, IgE, IgD or IgM. The Fc region is a C-terminal region of an Igheavy chain, which is responsible for binding to Fc receptors that carryout activities such as recycling (which results in prolonged half-life),antibody dependent cell-mediated cytotoxicity (ADCC), and complementdependent cytotoxicity (CDC).

For example, according to some definitions the human IgG heavy chain Fcregion stretches from Cys226 to the C-terminus of the heavy chain. The“hinge region” generally extends from Glu216 to Pro230 of human IgG1(hinge regions of other IgG isotypes may be aligned with the IgG1sequence by aligning the cysteines involved in cysteine bonding). The Fcregion of an IgG includes two constant domains, CH2 and CH3. The CH2domain of a human IgG Fc region usually extends from amino acids 231 toamino acid 341. The CH3 domain of a human IgG Fc region usually extendsfrom amino acids 342 to 447. References made to amino acid numbering ofimmunoglobulins or immunoglobulin fragments, or regions, are all basedon Kabat et al. 1991, Sequences of Proteins of Immunological Interest,U.S. Department of Public Health, Bethesda, Md., incorporated herein byreference. In related embodiments, the Fc region may comprise one ormore native or modified constant regions from an immunoglobulin heavychain, other than CH1, for example, the CH2 and CH3 regions of IgG andIgA, or the CH3 and CH4 regions of IgE.

Suitable conjugate moieties include portions of immunoglobulin sequencethat include the FcRn binding site. FcRn, a salvage receptor, isresponsible for recycling immunoglobulins and returning them tocirculation in the blood. The region of the Fc portion of IgG that bindsto the FcRn receptor has been described based on X-ray crystallography(Burmeister et al. 1994, Nature 372:379). The major contact area of theFc with the FcRn is near the junction of the CH2 and CH3 domains.Fc-FcRn contacts are all within a single Ig heavy chain. The majorcontact sites include amino acid residues 248, 250-257, 272, 285, 288,290-291, 308-311, and 314 of the CH2 domain and amino acid residues385-387, 428, and 433-436 of the CH3 domain.

Some conjugate moieties may or may not include FcγR binding site(s).FcγR are responsible for antibody-dependent cell-mediated cytotoxicity(ADCC) and complement-dependent cytotoxicity (CDC). Examples ofpositions within the Fc region that make a direct contact with FcγR areamino acids 234-239 (lower hinge region), amino acids 265-269 (B/Cloop), amino acids 297-299 (C′/E loop), and amino acids 327-332 (F/G)loop (Sondermann et al., Nature 406: 267-273, 2000). The lower hingeregion of IgE has also been implicated in the FcRI binding (Henry, etal., Biochemistry 36, 15568-15578, 1997). Residues involved in IgAreceptor binding are described in Lewis et al., (J Immunol.175:6694-701, 2005). Amino acid residues involved in IgE receptorbinding are described in Sayers et al. (J Biol Chem. 279(34):35320-5,2004).

Amino acid modifications may be made to the Fc region of animmunoglobulin. Such variant Fc regions comprise at least one amino acidmodification in the CH3 domain of the Fc region (residues 342-447)and/or at least one amino acid modification in the CH2 domain of the Fcregion (residues 231-341). Mutations believed to impart an increasedaffinity for FcRn include T256A, T307A, E380A, and N434A (Shields et al.2001, J. Biol. Chem. 276:6591). Other mutations may reduce binding ofthe Fc region to FcγRI, FcγRIIA, FcγRIIB, and/or FcγRIIIA withoutsignificantly reducing affinity for FcRn. For example, substitution ofthe Asn at position 297 of the Fc region with Ala or another amino acidremoves a highly conserved N-glycosylation site and may result inreduced immunogenicity with concomitant prolonged half-life of the Fcregion, as well as reduced binding to FcγRs (Routledge et al. 1995,Transplantation 60:847; Friend et al. 1999, Transplantation 68:1632;Shields et al. 1995, J. Biol. Chem. 276:6591). Amino acid modificationsat positions 233-236 of IgG1 have been made that reduce binding to FcγRs(Ward and Ghetie 1995, Therapeutic Immunology 2:77 and Armour et al.1999, Eur. J. Immunol. 29:2613). Some exemplary amino acid substitutionsare described in U.S. Pat. Nos. 7,355,008 and 7,381,408, each of whichis incorporated by reference herein in its entirety.

In some embodiments, the binding agent is fused to alkaline phosphatase(AP). Methods for making Fc or AP fusion agents are provided in WO02/060950.

Chimeric Antigen Receptors (CARs)

In exemplary aspects, the effector domain is a T-cell signaling domain.In exemplary aspects, the conjugate is a chimeric antigen receptor(CAR). Chimeric antigen receptors (CARs) are engineered transmembraneproteins that combine the specificity of an antigen-specific antibodywith a T-cell receptor's function. In general, CARs comprise anectodomain, a spacer region, a transmembrane domain, and an endodomain.The ectodomain of a CAR in exemplary aspects comprises an antigenrecognition region, which may be an scFV of an antigen-specificantibody. The ectodomain also in some embodiments comprises a signalpeptide which directs the nascent protein into the endoplasmicreticulum. In exemplary aspects, the ectodomain comprises a spacer whichlinks the antigen recognition region to the transmembrane domain. Thetransmembrane (TM) domain is the portion of the CAR which traverses thecell membrane. In exemplary aspects, the TM domain comprises ahydrophobic alpha helix. In exemplary aspects, the TM domain comprisesall or a portion of the TM domain of CD28. In exemplary aspects, the TMdomain comprises all or a portion of the TM domain of CD8α. Theendodomain of a CAR comprises one or more signaling domains. Inexemplary aspects, the endodomain comprises the zeta chain of CD3, whichcomprises three copies of the Immunoreceptor Tyrosine-based ActivationMotif (ITAM). An ITAM generally comprises a Tyr residue separated by twoamino acids from a Leu or Ile. In the case of immune cell receptors,e.g., the T cell receptor and the B cell receptor, the ITAMs occur inmultiples (at least two) and each ITAM is separated from another by 6-8amino acids. The endodomain of CARs may also comprises additionalsignaling domains, e.g., portions of proteins that are important fordownstream signal transduction. In exemplary aspects, the endodomaincomprises signaling domains from one or more of CD28, 41BB or 4-1BB(CD137), ICOS, CD27, CD40, OX40 (CD134), or Myd88. Sequences encodingsignaling domains of such proteins are provided herein as SEQ ID NOs:39-42, 68-79, 81, and 83. Methods of making CARs, expressing them incells, e.g., T-cells, and utilizing the CAR-expressing T-cells intherapy, are known in the art. See, e.g., International PatentApplication Publication Nos. WO2014/208760, WO2014/190273,WO2014/186469, WO2014/184143, WO2014180306, WO2014/179759,WO2014/153270, U.S. Application Publication Nos. US20140369977,US20140322212, US20140322275, US20140322183, US20140301993,US20140286973, US20140271582, US20140271635, US20140274909, EuropeanApplication Publication No. 2814846, each of which are incorporated byreference in their entirety.

In exemplary aspects, the conjugate of the disclosure is anIL13Rα2-specific chimeric antigen receptor (CAR) comprising a bindingagent described herein, a hinge region, and an endodomain comprising asignaling domain of a CD3 zeta chain and a signaling domain of CD28,CD134, and/or CD137. In exemplary aspects, the CAR comprises (A) each ofthe amino acid sequence of: NYLMN (SEQ ID NO: 1); RIDPYDGDIDYNQNFKD (SEQID NO: 2); GYGTAYGVDY (SEQ ID NO: 3); RASESVDNYGISFMN (SEQ ID NO: 4);AASRQGSG (SEQ ID NO: 5); and QQSKEVPWT (SEQ ID NO: 6), (B) a hingeregion; and (C) an endodomain comprising a signaling domain of a CD3zeta chain and a signaling domain of CD28, CD134, and/or CD137. Inexemplary aspects, the CD3 zeta chain signaling domain comprises theamino acid sequence of SEQ ID NO: 41. In exemplary aspects, the CARfurther comprises a transmembrane (TM) domain based on the TM domain ofCD28. In exemplary aspects, the CAR comprises the amino acid sequence ofSEQ ID NO: 47. In exemplary aspects, the CAR further comprises atransmembrane (TM) domain based on the TM domain of CD8α. In exemplaryaspects, the CAR comprises the amino acid sequence of SEQ ID NO: 85. Inexemplary aspects, the hinge region comprises the amino acid sequence ofSEQ ID NO: 35 or SEQ ID NO: 37. In exemplary aspects, the CAR comprisesthe amino acid sequence of SEQ ID NO: 49 or SEQ ID NO: 51. In exemplaryaspects, the endodomain of the CAR of the disclosures comprises theamino acid sequence of SEQ ID NO: 53 or SEQ ID NO: 55. In exemplaryaspects, the endodomain of the CAR of the disclosure comprises the aminoacid sequence of SEQ ID NO: 87 or SEQ ID NO: 89. In exemplary aspects,the endodomain of the CAR of the disclosure comprises the amino acidsequence of SEQ ID NO: 91, SEQ ID NO: 93 or SEQ ID NO: 95. In exemplaryaspects, the endodomain of the CAR of the disclosure comprises the aminoacid sequence of SEQ ID NO: 97, SEQ ID NO: 99 or SEQ ID NO: 101.

In exemplary aspects, the endodomain further comprises a signalingdomain of one or more of: CD137, CD134, CD27, CD40, ICOS, and Myd88. Inexemplary aspects, the endodomain comprises one or more of the aminoacid sequences of SEQ ID NOs: 68, 70, 72, 74, 76, and 78, which providea sequence comprising a CD27 signaling domain, a sequence comprising aCD40 signaling domain, a sequence comprising a CD134 signaling domain, asequence comprising a CD137 signaling domain, a sequence comprising anICOS signaling domain, and a sequence comprising a Myd88 signalingdomain, respectively.

In exemplary aspects, the CAR comprises (A) each of the amino acidsequence of: NYLMN (SEQ ID NO: 1); RIDPYDGDIDYNQNFKD (SEQ ID NO: 2);GYGTAYGVDY (SEQ ID NO: 3); RASESVDNYGISFMN (SEQ ID NO: 4); AASRQGSG (SEQID NO: 5); and QQSKEVPWT (SEQ ID NO: 6), (B) a hinge region; (C) anendodomain comprising a signaling domain of a CD3 zeta chain and asignaling domain of CD28 and at least one other signaling domain. Inexemplary aspects, the CAR comprises an endodomain comprising asignaling domain of 41BB (CD137). In exemplary aspects the CAR comprisesan endodomain comprising an amino acid sequence of SEQ ID NO: 81. Inexemplary aspects, the CD137 signaling is N-terminal to a CD3 zeta chainsignaling chain. In exemplary aspects, the endodomain comprises theamino acid sequence of SEQ ID NO: 87. In exemplary aspects, the CARcomprises the amino acid sequence of SEQ ID NO: 91. In exemplaryaspects, the CAR comprises the amino acid sequence of SEQ ID NO: 97.

In exemplary aspects, the CAR comprises an endodomain comprising asignaling domain of OX40 (CD134). In exemplary aspects the CAR comprisesan endodomain comprising an amino acid sequence of SEQ ID NO: 83. Inexemplary aspects, the CD137 signaling is N-terminal to a CD3 zeta chainsignaling chain. In exemplary aspects, the endodomain comprises theamino acid sequence of SEQ ID NO: 89. In exemplary aspects, the CARcomprises the amino acid sequence of SEQ ID NO: 95. In exemplaryaspects, the CAR comprises the amino acid sequence of SEQ ID NO: 99.

In exemplary aspects, the CAR comprises (A) each of the amino acidsequence of: NYLMN (SEQ ID NO: 1); RIDPYDGDIDYNQNFKD (SEQ ID NO: 2);GYGTAYGVDY (SEQ ID NO: 3); RASESVDNYGISFMN (SEQ ID NO: 4); AASRQGSG (SEQID NO: 5); and QQSKEVPWT (SEQ ID NO: 6), (B) a hinge region; (C) atransmembrane domain of CD8a chain, and (D) an endodomain comprising asignaling domain of a CD3 zeta chain, and, optionally, at least oneother signaling domain. In exemplary aspects, the transmembrane domaincomprises the amino acid sequence of SEQ ID NO: 85. In exemplaryaspects, the CAR further comprises a CD137 signaling domain and a CD3zeta chain signaling domain. In exemplary aspects, the CAR comprises theamino acid sequence of SEQ ID NO: 93. In exemplary aspects, the CARcomprises the amino acid sequence of SEQ ID NO: 101.

As an example, sequences of three additional IL13Rα2-specific CARs areprovided. One CAR contains a CD8a TM domain, and a 41BB.zeta signalingdomain (SEQ ID NO:93 encoded by SEQ ID NO:94). The other two CARscontain a CD28 TM domain and either a CD28.CD134.zeta (SEQ ID NO:99encoded by SEQ ID NO:100) or CD28.CD137.zeta (SEQ ID NO:101 encoded bySEQ ID NO:102) signaling domain.

Nucleic Acids, Vectors, Host Cells

Further provided by the disclosures is a nucleic acid comprising anucleotide sequence encoding any of the binding agents and conjugates(e.g., chimeric proteins, fusion proteins, CARs) described herein. Thenucleic acid may comprise any nucleotide sequence which encodes any ofthe binding agents and conjugates described herein. In exemplaryaspects, the nucleic acid comprises a nucleotide sequence encoding eachof the CDRs of SEQ ID NOs: 1-6. In exemplary aspects, the nucleic acidof the disclosures comprises a nucleic acid sequence which encodes a SEQID NO: 7 and/or SEQ ID NO: 8. In exemplary aspects, the nucleic acid ofthe disclosures comprises a nucleic acid sequence which encodes SEQ IDNO: 13 or SEQ ID NO: 14. In exemplary aspects, the nucleic acid providedherein comprises the sequence of SEQ ID NO: 15 and/or SEQ ID NO: 16. Inexemplary aspects, the nucleic acid comprises a nucleotide sequence ofSEQ ID NO: 66 or 67. In exemplary aspects, the nucleic acid comprises anucleotide sequence which encodes the sequence of SEQ I DNO: 25. Inexemplary aspects, the nucleic acid comprises a nucleotide sequence ofSEQ ID NO: 26. In exemplary aspects, the nucleic acid comprises anucleotide sequence which encodes each of SEQ ID NOs: 1-6 and encodesSEQ ID NO: 28 or 30. In exemplary aspects, the nucleic acid comprises anucleotide sequence which encodes each of SEQ ID NOs: 1-6 and encodes anamino acid sequence which is at least 90% identical to SEQ ID NO: 28 or30. In exemplary aspects, the nucleic acid comprises a nucleotidesequence which encodes each of SEQ ID NOs: 1-6 and comprises SEQ ID NO:29 or 31. In exemplary aspects, the nucleic acid comprises a nucleotidesequence which encodes SEQ ID NO: 33. In exemplary aspects, the nucleicacid comprises a nucleotide sequence of SEQ ID NO: 34. In exemplaryaspects, the nucleic acid comprises a nucleotide sequence which encodeseach of SEQ ID NOs: 1-6 and encodes SEQ ID NO: 35 or 37. In exemplaryaspects, the nucleic acid comprises a nucleotide sequence which encodeseach of SEQ ID NOs: 1-6 and comprises SEQ ID NO: 36 or 38. In exemplaryaspects, the nucleic acid comprises a nucleotide sequence which encodeseach of SEQ ID NOs: 1-6 and encodes SEQ ID NO: 39 or 41. In exemplaryaspects, the nucleic acid comprises a nucleotide sequence which encodeseach of SEQ ID NOs: 1-6 and comprises SEQ ID NO: 40 or 42. In exemplaryaspects, the nucleic acid comprises a nucleotide sequence which encodeseach of SEQ ID NOs: 1-6 and encodes SEQ ID NO: 47. In exemplary aspects,the nucleic acid comprises a nucleotide sequence which encodes each ofSEQ ID NOs: 1-6 and comprises SEQ ID NO: 48. In exemplary aspects, thenucleic acid comprises a nucleotide sequence which encodes each of SEQID NOs: 1-6 and encodes SEQ ID NO: 49 or 51. In exemplary aspects, thenucleic acid comprises a nucleotide sequence which encodes each of SEQID NOs: 1-6 and comprises SEQ ID NO: 50 or 52. In exemplary aspects, thenucleic acid comprises a nucleotide sequence which encodes SEQ ID NO: 53or 55. In exemplary aspects, the nucleic acid comprises a nucleotidesequence which encodes each of SEQ ID NOs: 1-6 and comprises SEQ ID NO:54 or 56. In exemplary aspects, the nucleic acid comprises a nucleotidesequence which encodes each of SEQ ID NOs: 1-6 and encodes one or moreof SEQ ID NOs: 68, 70, 72, 74, 76, and 78. In exemplary aspects, thenucleic acid comprises a nucleotide sequence which encodes each of SEQID NOs: 1-6 and comprises one or more of SEQ ID NOs: 69, 71, 73, 75, 77,and 79. In exemplary aspects, the nucleic acid comprises a nucleotidesequence which encodes each of SEQ ID NOs: 1-6 and comprises one or moreof SEQ ID NOs: 82, 84, 86, 88, 90, 92, 94, 96. In exemplary aspects, thenucleic acid comprises a nucleotide sequence comprising one of SEQ IDNOs: 98, 100, and 102.

By “nucleic acid” as used herein includes “polynucleotide,”“oligonucleotide,” and “nucleic acid molecule,” and generally means apolymer of DNA or RNA, which may be single-stranded or double-stranded,synthesized or obtained (e.g., isolated and/or purified) from naturalsources, which may contain natural, non-natural or altered nucleotides,and which may contain a natural, non-natural or altered internucleotidelinkage, such as a phosphoroamidate linkage or a phosphorothioatelinkage, instead of the phosphodiester found between the nucleotides ofan unmodified oligonucleotide. It is generally preferred that thenucleic acid does not comprise any insertions, deletions, inversions,and/or substitutions. However, it may be suitable in some instances, asdiscussed herein, for the nucleic acid to comprise one or moreinsertions, deletions, inversions, and/or substitutions.

In exemplary aspects, the nucleic acids of the disclosures arerecombinant. As used herein, the term “recombinant” refers to (i)molecules that are constructed outside living cells by joining naturalor synthetic nucleic acid segments to nucleic acid molecules that mayreplicate in a living cell, or (ii) molecules that result from thereplication of those described in (i) above. For purposes herein, thereplication may be in vitro replication or in vivo replication.

The nucleic acids in exemplary aspects are constructed based on chemicalsynthesis and/or enzymatic ligation reactions using procedures known inthe art. See, for example, Sambrook et al., supra, and Ausubel et al.,supra. For example, a nucleic acid may be chemically synthesized usingnaturally occurring nucleotides or variously modified nucleotidesdesigned to increase the biological stability of the molecules or toincrease the physical stability of the duplex formed upon hybridization(e.g., phosphorothioate derivatives and acridine substitutednucleotides). Examples of modified nucleotides that may be used togenerate the nucleic acids include, but are not limited to,5-fluorouracil, 5-bromouracil, 5-chIorouracil, 5-iodouracil,hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridme,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N-substitutedadenine, 7-methylguanine, 5-methylammomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine. Alternatively, one or more of the nucleicacids of the disclosures may be purchased from companies, such asMacromolecular Resources (Fort Collins, Colo.) and Synthegen (Houston,Tex.).

The nucleic acids of the disclosures in exemplary aspects areincorporated into a recombinant expression vector. In this regard, thedisclosures provides recombinant expression vectors comprising any ofthe nucleic acids of the disclosures. For purposes herein, the term“recombinant expression vector” means a genetically-modifiedoligonucleotide or polynucleotide construct that permits the expressionof an mRNA, protein, polypeptide, or peptide by a host cell, when theconstruct comprises a nucleotide sequence encoding the mRNA, protein,polypeptide, or peptide, and the vector is contacted with the cell underconditions sufficient to have the mRNA, protein, polypeptide, or peptideexpressed within the cell. The vectors of the disclosures are notnaturally-occurring as a whole. However, parts of the vectors may benaturally-occurring. The inventive recombinant expression vectors maycomprise any type of nucleotides, including, but not limited to DNA andRNA, which may be single-stranded or double-stranded, synthesized orobtained in part from natural sources, and which may contain natural,non-natural or altered nucleotides. The recombinant expression vectorsmay comprise naturally-occurring or non-naturally occurringinternucleotide linkages, or both types of linkages. In exemplaryaspects, the altered nucleotides or non-naturally occurringinternucleotide linkages do not hinder the transcription or replicationof the vector.

The recombinant expression vector of the disclosures may be any suitablerecombinant expression vector, and may be used to transform or transfectany suitable host. Suitable vectors include those designed forpropagation and expansion or for expression or both, such as plasmidsand viruses. The vector may be selected from the group consisting of thepUC series (Fermentas Life Sciences), the pBluescript series(Stratagene, LaJolla, Calif.), the pET series (Novagen, Madison, Wis.),the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEX series(Clontech, Palo Alto, Calif.). Bacteriophage vectors, such as λGTIO,λGT1 1, λZapII (Stratagene), λEMBL4, and λNM1 149, also may be used.Examples of plant expression vectors include pBIO1, pBI101.2, pBI101.3,pBI121 and pBIN19 (Clontech). Examples of animal expression vectorsinclude pEUK-Cl, pMAM and pMAMneo (Clontech). Preferably, therecombinant expression vector is a viral vector, e.g., a retroviralvector.

The recombinant expression vectors of the disclosures may be preparedusing standard recombinant DNA techniques described in, for example,Sambrook et al., supra, and Ausubel et al., supra. Constructs ofexpression vectors, which are circular or linear, may be prepared tocontain a replication system functional in a prokaryotic or eukaryotichost cell. Replication systems may be derived, e.g., from CoIE1, 2μplasmid, λ, SV40, bovine papilloma virus, and the like.

In exemplary aspects, the recombinant expression vector comprisesregulatory sequences, such as transcription and translation initiationand termination codons, which are specific to the type of host (e.g.,bacterium, fungus, plant, or animal) into which the vector is to beintroduced, as appropriate and taking into consideration whether thevector is DNA- or RNA-based.

The recombinant expression vector may include one or more marker genes,which allow for selection of transformed or transfected hosts. Markergenes include biocide resistance, e.g., resistance to antibiotics, heavymetals, etc., complementation in an auxotrophic host to provideprototrophy, and the like. Suitable marker genes for the inventiveexpression vectors include, for instance, neomycin/G418 resistancegenes, hygromycin resistance genes, histidinol resistance genes,tetracycline resistance genes, and ampicillin resistance genes.

The recombinant expression vector may comprise a native or normativepromoter operably linked to the nucleotide sequence encoding the bindingagent or conjugate or to the nucleotide sequence which is complementaryto or which hybridizes to the nucleotide sequence encoding the bindingagent or conjugate. The selection of promoters, e.g., strong, weak,inducible, tissue-specific and developmental-specific, is within theordinary skill of the artisan.

Similarly, the combining of a nucleotide sequence with a promoter isalso within the skill of the artisan. The promoter may be a non-viralpromoter or a viral promoter, e.g., a cytomegalovirus (CMV) promoter, anSV40 promoter, an RSV promoter, and a promoter found in thelong-terminal repeat of the murine stem cell virus.

The inventive recombinant expression vectors may be designed for eithertransient expression, for stable expression, or for both. Also, therecombinant expression vectors may be made for constitutive expressionor for inducible expression. Further, the recombinant expression vectorsmay be made to include a suicide gene.

As used herein, the term “suicide gene” refers to a gene that causes thecell expressing the suicide gene to die. The suicide gene may be a genethat confers sensitivity to an agent, e.g., a drug, upon the cell inwhich the gene is expressed, and causes the cell to die when the cell iscontacted with or exposed to the agent. Suicide genes are known in theart (see, for example, Suicide Gene Therapy: Methods and Reviews.Springer, Caroline J. (Maycer Research UK Centre for Maycer Therapeuticsat the Institute of Maycer Research, Sutton, Surrey, UK), Humana Press,2004) and include, for example, the Herpes Simplex Virus (HSV) thymidinekinase (TK) gene, cytosine deaminase, purine nucleoside phosphorylase,and nitroreductase.

The disclosures further provides a host cell comprising any of thenucleic acids or vectors described herein. As used herein, the term“host cell” refers to any type of cell that may contain the nucleic acidor vector described herein. In exemplary aspects, the host cell is aeukaryotic cell, e.g., plant, animal, fungi, or algae, or may be aprokaryotic cell, e.g., bacteria or protozoa. In exemplary aspects, thehost cells is a cell originating or obtained from a subject, asdescribed herein. In exemplary aspects, the host cell originates from oris obtained from a mammal. As used herein, the term “mammal” refers toany mammal, including, but not limited to, mammals of the orderRodentia, such as mice and hamsters, and mammals of the orderLagomorpha, such as rabbits. It is preferred that the mammals are fromthe order Carnivora, including Felines (cats) and Canines (dogs). It ismore preferred that the mammals are from the order Artiodactyla,including bovines (cows) and swines (pigs) or of the orderPerssodactyla, including equines (horses). It is most preferred that themammals are of the order Primates, Ceboids, or Simoids (monkeys) or ofthe order Anthropoids (humans and apes). An especially preferred mammalis the human.

In exemplary aspects, the host cell is a cultured cell or a primarycell, i.e., isolated directly from an organism, e.g., a human. The hostcell in exemplary aspects is an adherent cell or a suspended cell, i.e.,a cell that grows in suspension. Suitable host cells are known in theart and include, for instance, DH5α E. coli cells, Chinese hamsterovarian (CHO) cells, monkey VERO cells, T293 cells, COS cells, HEK293cells, and the like. For purposes of amplifying or replicating therecombinant expression vector, the host cell is preferably a prokaryoticcell, e.g., a DH5α cell. For purposes of producing a binding agent or aconjugate, the host cell is in some aspects a mammalian cell. Inexemplary aspects, the host cell is a human cell. While the host cellmay be of any cell type, the host cell may originate from any type oftissue, and may be of any developmental stage. In exemplary aspects, thehost cell is a hematopoietic stem cell or progenitor cell. See, e.g,Nakamura De Oliveira et al., Human Gene Therapy 24:824-839 (2013). Thehost cell in exemplary aspects is a peripheral blood lymphocyte (PBL).In exemplary aspects, the host cell is a natural killer cell. Inexemplary aspects, the host cell is a T cell.

For purposes herein, the T cell may be any T cell, such as a cultured Tcell, e.g., a primary T cell, or a T cell from a cultured T cell line,e.g., Jurkat, SupTl, etc., or a T cell obtained from a mammal. Ifobtained from a mammal, the T cell may be obtained from numeroussources, including but not limited to blood, bone marrow, lymph node,the thymus, or other tissues or fluids. T cells may also be enriched foror purified. The T cell may be obtained by maturing hematopoietic stemcells, either in vitro or in vivo, into T cells. In exemplary aspects,the T cell is a human T cell. In exemplary aspects, the T cell is a Tcell isolated from a human. The T cell may be any type of T cell,including NKT cell, and may be of any developmental stage, including butnot limited to, CD4+/CD8+ double positive T cells, CDA+ helper T cells,e.g., Th1 and Th2 cells, CD8+ T cells (e.g., cytotoxic T cells),peripheral blood mononuclear cells (PBMCs), peripheral blood leukocytes(PBLs), tumor infiltrating cells (TILs), memory T cells, naive T cells,and the like. Preferably, the T cell is a CD8+ T cell or a CD4+ T cell.

Also provided by the disclosures is a population of cells comprising atleast one host cell described herein. The population of cells may be aheterogeneous population comprising the host cell comprising any of therecombinant expression vectors described, in addition to at least oneother cell, e.g., a host cell (e.g., a T cell), which does not compriseany of the recombinant expression vectors, or a cell other than a Tcell, e.g., a B cell, a macrophage, a neutrophil, an erythrocyte, ahepatocyte, an endothelial cell, an epithelial cells, a muscle cell, abrain cell, etc. Alternatively, the population of cells may be asubstantially homogeneous population, in which the population comprisesmainly of host cells (e.g., consisting essentially of) comprising therecombinant expression vector. The population also may be a clonalpopulation of cells, in which all cells of the population are clones ofa single host cell comprising a recombinant expression vector, such thatall cells of the population comprise the recombinant expression vector.In exemplary embodiments of the disclosures, the population of cells isa clonal population comprising host cells expressing a nucleic acid or avector described herein.

Pharmaceutical Compositions and Routes of Administration

In some embodiments of the disclosures, the binding agents, conjugates,nucleic acids, vectors, host cells, or populations of cells, are admixedwith a pharmaceutically acceptable carrier. Accordingly, pharmaceuticalcompositions comprising any of the binding agents, conjugates, nucleicacids, vectors, host cells, or populations of cells described herein andcomprising a pharmaceutically acceptable carrier, diluent, or excipientare contemplated.

The pharmaceutically acceptable carrier is any of those conventionallyused and is limited only by physico-chemical considerations, such assolubility and lack of reactivity with the active binding agent(s), andby the route of administration. The pharmaceutically acceptable carriersdescribed herein, for example, vehicles, adjuvants, excipients, anddiluents, are well-known to those skilled in the art and are readilyavailable to the public. In one aspect the pharmaceutically acceptablecarrier is one that is chemically inert to the active ingredient(s) ofthe pharmaceutical composition, e.g., the first binding agent and thesecond binding agent, and one which has no detrimental side effects ortoxicity under the conditions of use. The carrier in some embodimentsdoes not produce adverse, allergic, or other untoward reactions whenadministered to an animal or a human. The pharmaceutical composition insome aspects is free of pyrogens, as well as other impurities that couldbe harmful to humans or animals. Pharmaceutically acceptable carriersinclude any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents and thelike; the use of which are well known in the art.

Acceptable carriers, excipients or stabilizers are nontoxic torecipients and are preferably inert at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, or otherorganic acids; antioxidants such as ascorbic acid; low molecular weightpolypeptides; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, arginine or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugaralcohols such as mannitol or sorbitol; salt-forming counterions such assodium; and/or nonionic surfactants such as Tween, Pluronics orpolyethylene glycol (PEG).

Therapeutic formulations of the compositions useful for practicing themethods disclosed herein, such as polypeptides, polynucleotides, orantibodies, may be prepared for storage by mixing the selectedcomposition having the desired degree of purity with optionalphysiologically pharmaceutically-acceptable carriers, excipients, orstabilizers (Remington's Pharmaceutical Sciences, 18th edition, A. R.Gennaro, ed., Mack Publishing Company (1990)) in the form of alyophilized cake or an aqueous solution. Pharmaceutical compositions maybe produced by admixing with one or more suitable carriers or adjuvantssuch as water, mineral oil, polyethylene glycol, starch, talcum,lactose, thickeners, stabilizers, suspending agents, and the like. Suchcompositions may be in the form of solutions, suspensions, tablets,capsules, creams, salves, ointments, or other conventional forms.

The composition to be used for in vivo administration should be sterile.This is readily accomplished by filtration through sterile filtrationmembranes, prior to or following lyophilization and reconstitution.Therapeutic compositions generally are placed into a container having asterile access port, for example, an intravenous solution bag or vialhaving a stopper pierceable by a hypodermic injection needle. Thepharmaceutical forms suitable for injectable use include sterile aqueoussolutions or dispersions and sterile powders for the extemporaneouspreparation of sterile injectable solutions or dispersions. In somecases the form should be sterile and should be fluid to the extent thateasy syringability exists. It should be stable under the conditions ofmanufacture and storage and should be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Thecomposition for parenteral administration ordinarily will be stored inlyophilized form or in solution.

The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin, by the maintenanceof the required particle size in the case of dispersion, and by the useof surfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and/or antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the inclusion in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

The choice of carrier will be determined in part by the particular typeof binding agents of the pharmaceutical composition, as well as by theparticular route used to administer the pharmaceutical composition.Accordingly, there are a variety of suitable formulations of thepharmaceutical composition.

The pharmaceutical composition of the present disclosures can compriseany pharmaceutically acceptable ingredient including, for example,acidifying agents, additives, adsorbents, aerosol propellants, airdisplacement agents, alkalizing agents, anticaking agents,anticoagulants, antimicrobial preservatives, antioxidants, antiseptics,bases, binders, buffering agents, chelating agents, coating agents,coloring agents, desiccants, detergents, diluents, disinfectants,disintegrants, dispersing agents, dissolution-enhancing agents, dyes,emollients, emulsifying agents, emulsion stabilizers, fillers,film-forming agents, flavor enhancers, flavoring agents, flow enhancers,gelling agents, granulating agents, humectants, lubricants,mucoadhesives, ointment bases, ointments, oleaginous vehicles, organicbases, pastille bases, pigments, plasticizers, polishing agents,preservatives, sequestering agents, skin penetrants, solubilizingagents, solvents, stabilizing agents, suppository bases, surface activeagents, surfactants, suspending agents, sweetening agents, therapeuticagents, thickening agents, tonicity agents, toxicity agents,viscosity-increasing agents, water-absorbing agents, water-misciblecosolvents, water softeners, or wetting agents.

The pharmaceutical compositions may be formulated to achieve aphysiologically compatible pH. In some embodiments, the pH of thepharmaceutical composition may be at least 5, at least 5.5, at least 6,at least 6.5, at least 7, at least 7.5, at least 8, at least 8.5, atleast 9, at least 9.5, at least 10, or at least 10.5 up to and includingpH 11, depending on the formulation and route of administration. Incertain embodiments, the pharmaceutical compositions may comprisebuffering agents to achieve a physiologically compatible pH. Thebuffering agents may include any compounds capable of buffering at thedesired pH such as, for example, phosphate buffers (e.g., PBS),triethanolamine, Tris, bicine, TAPS, tricine, HEPES, TES, MOPS, PIPES,cacodylate, MES, and others known in the art.

In some embodiments, the pharmaceutical composition comprising thebinding agents described herein is formulated for parenteraladministration, subcutaneous administration, intravenous administration,intramuscular administration, intraarterial administration, intrathecaladministration, or interperitoneal administration. In other embodiments,the pharmaceutical composition is administered via nasal, spray, oral,aerosol, rectal, or vaginal administration. The compositions may beadministered by infusion, bolus injection or by implantation device.

The following discussion on routes of administration is merely providedto illustrate exemplary embodiments and should not be construed aslimiting the scope of the disclosed subject matter in any way.

Formulations suitable for oral administration can consist of (a) liquidsolutions, such as an effective amount of the composition of the presentdisclosure dissolved in diluents, such as water, saline, or orangejuice; (b) capsules, sachets, tablets, lozenges, and troches, eachcontaining a predetermined amount of the active ingredient, as solids orgranules; (c) powders; (d) suspensions in an appropriate liquid; and (e)suitable emulsions. Liquid formulations may include diluents, such aswater and alcohols, for example, ethanol, benzyl alcohol, and thepolyethylene alcohols, either with or without the addition of apharmaceutically acceptable surfactant. Capsule forms can be of theordinary hard- or soft-shelled gelatin type containing, for example,surfactants, lubricants, and inert fillers, such as lactose, sucrose,calcium phosphate, and corn starch. Tablet forms can include one or moreof lactose, sucrose, mannitol, corn starch, potato starch, alginic acid,microcrystalline cellulose, acacia, gelatin, guar gum, colloidal silicondioxide, croscarmellose sodium, talc, magnesium stearate, calciumstearate, zinc stearate, stearic acid, and other excipients, colorants,diluents, buffering agents, disintegrating agents, moistening agents,preservatives, flavoring agents, and other pharmacologically compatibleexcipients. Lozenge forms can comprise a composition of the disclosurein a flavor, usually sucrose and acacia or tragacanth, as well aspastilles comprising a composition of the disclosure in an inert base,such as gelatin and glycerin, or sucrose and acacia, emulsions, gels,and the like, optionally also containing such excipients as are known inthe art.

The compositions of the disclosure, alone or in combination with othersuitable components, can be delivered via pulmonary administration andcan be made into aerosol formulations to be administered via inhalation.These aerosol formulations can be placed into pressurized acceptablepropellants, such as dichlorodifluoromethane, propane, nitrogen, and thelike. They also may be formulated as pharmaceuticals for non-pressuredpreparations, such as in a nebulizer or an atomizer. Such sprayformulations also may be used to spray mucosa. In some embodiments, thecomposition is formulated into a powder blend or into microparticles ornanoparticles. Suitable pulmonary formulations are known in the art.See, e.g., Qian et al., Int J Pharm 366: 218-220 (2009); Adjei andGarren, Pharmaceutical Research, 7(6): 565-569 (1990); Kawashima et al.,J Controlled Release 62(1-2): 279-287 (1999); Liu et al., Pharm Res10(2): 228-232 (1993); International Patent Application Publication Nos.WO 2007/133747 and WO 2007/141411.

Topical formulations are well-known to those of skill in the art. Suchformulations are particularly suitable in the context of the inventionfor application to the skin.

In some embodiments, the pharmaceutical composition described herein isformulated for parenteral administration. For purposes herein,parenteral administration includes, but is not limited to, intravenous,intraarterial, intramuscular, intracerebral, intracerebroventricular,intracardiac, subcutaneous, intraosseous, intradermal, intrathecal,intraperitoneal, retrobulbar, intrapulmonary, intravesical, andintracavernosal injections or infusions. Administration by surgicalimplantation at a particular site is contemplated as well.

Formulations suitable for parenteral administration include aqueous andnon-aqueous, isotonic sterile injection solutions, which can containanti-oxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The term, “parenteral” means not through the alimentary canal but bysome other route such as subcutaneous, intramuscular, intraspinal, orintravenous. The composition of the present disclosure can beadministered with a physiologically acceptable diluent in apharmaceutical carrier, such as a sterile liquid or mixture of liquids,including water, saline, aqueous dextrose and related sugar solutions,an alcohol, such as ethanol or hexadecyl alcohol, a glycol, such aspropylene glycol or polyethylene glycol, dimethylsulfoxide, glycerol,ketals such as 2,2-dimethyl-1,5,3-dioxolane-4-methanol, ethers,poly(ethyleneglycol) 400, oils, fatty acids, fatty acid esters orglycerides, or acetylated fatty acid glycerides with or without theaddition of a pharmaceutically acceptable surfactant, such as a soap ora detergent, a suspending agent, such as pectin, carbomers,methylcellulose, hydroxypropylmethylcellulose, orcarboxymethylcellulose, or emulsifying agents and other pharmaceuticaladjuvants.

Oils, which can be used in parenteral formulations include petroleum,animal, vegetable, or synthetic oils. Specific examples of oils includepeanut, soybean, sesame, cottonseed, corn, olive, petrolatum, andmineral. Suitable fatty acids for use in parenteral formulations includeoleic acid, stearic acid, and isostearic acid. Ethyl oleate andisopropyl myristate are examples of suitable fatty acid esters.

The parenteral formulations in some embodiments contain preservatives orbuffers. In order to minimize or eliminate irritation at the site ofinjection, such compositions optionally contain one or more nonionicsurfactants having a hydrophile-lipophile balance (HLB) of from about 12to about 17. The quantity of surfactant in such formulations willtypically range from about 5% to about 15% by weight. Suitablesurfactants include polyethylene glycol sorbitan fatty acid esters, suchas sorbitan monooleate and the high molecular weight adducts of ethyleneoxide with a hydrophobic base, formed by the condensation of propyleneoxide with propylene glycol. The parenteral formulations can bepresented in unit-dose or multi-dose sealed containers, such as ampoulesand vials, and can be stored in a freeze-dried (lyophilized) conditionrequiring only the addition of the sterile liquid excipient, forexample, water, for injections, immediately prior to use. Extemporaneousinjection solutions and suspensions can be prepared from sterilepowders, granules, and tablets of the kind previously described andknown in the art.

Injectable formulations are in accordance with the invention. Therequirements for effective pharmaceutical carriers for injectablecompositions are well-known to those of ordinary skill in the art (see,e.g., Pharmaceutics and Pharmacy Practice, J. B. Lippincott Company,Philadelphia, Pa., Banker and Chalmers, eds., pages 238-250 (1982), andASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages 622-630(1986)).

It will be appreciated by one of skill in the art that, in addition tothe above-described pharmaceutical compositions, the composition of thedisclosure can be formulated as inclusion complexes, such ascyclodextrin inclusion complexes, or liposomes.

Dose

For purposes herein, the amount or dose of the pharmaceuticalcomposition administered is sufficient to effect, e.g., a therapeutic orprophylactic response, in the subject or animal over a reasonable timeframe. For example, the dose of the pharmaceutical composition issufficient to treat or prevent a disease or medical condition in aperiod of from about 12 hours, about 18 hours, about 1 to 4 days orlonger, e.g., 5 days, 6 days, 1 week, 10 days, 2 weeks, 16 to 20 days,or more, from the time of administration. In certain embodiments, thetime period is even longer. The dose is determined by the efficacy ofthe particular pharmaceutical composition and the condition of theanimal (e.g., human), as well as the body weight of the animal (e.g.,human) to be treated.

Many assays for determining an administered dose are known in the art.In some embodiments, an assay which comprises comparing the extent towhich the binding agents block IL13Rα2-mediated cell growth uponadministration of a given dose to a mammal among a set of mammals eachof which is given a different dose of binding agents is used todetermine a starting dose to be administered to a mammal. The extent towhich the binding agents block IL13Rα2 mediated cell growth uponadministration of a certain dose can be assayed by methods known in theart.

The dose of the pharmaceutical composition also will be determined bythe existence, nature and extent of any adverse side effects that mightaccompany the administration of a particular pharmaceutical composition.Typically, the attending physician will decide the dosage of thepharmaceutical composition with which to treat each individual patient,taking into consideration a variety of factors, such as age, bodyweight, general health, diet, sex, binding agents of the pharmaceuticalcomposition to be administered, route of administration, and theseverity of the condition being treated.

By way of example and not intending to limit the invention, the dose ofthe binding agent of the present disclosure can be about 0.0001 to about1 g/kg body weight of the subject being treated/day, from about 0.0001to about 0.001 g/kg body weight/day, or about 0.01 mg to about 1 g/kgbody weight/day. The pharmaceutical composition in some aspects comprisethe binding agent of the present disclosure at a concentration of atleast A, wherein A is about 0.001 mg/ml, about 0.01 mg/ml, 0 about 1mg/ml, about 0.5 mg/ml, about 1 mg/ml, about 2 mg/ml, about 3 mg/ml,about 4 mg/ml, about 5 mg/ml, about 6 mg/ml, about 7 mg/ml, about 8mg/ml, about 9 mg/ml, about 10 mg/ml, about 11 mg/ml, about 12 mg/ml,about 13 mg/ml, about 14 mg/ml, about 15 mg/ml, about 16 mg/ml, about 17mg/ml, about 18 mg/ml, about 19 mg/ml, about 20 mg/ml, about 21 mg/ml,about 22 mg/ml, about 23 mg/ml, about 24 mg/ml, about 25 mg/ml orhigher. In some embodiments, the pharmaceutical composition comprisesthe binding agent at a concentration of at most B, wherein B is about 30mg/ml, about 25 mg/ml, about 24 mg/ml, about 23, mg/ml, about 22 mg/ml,about 21 mg/ml, about 20 mg/ml, about 19 mg/ml, about 18 mg/ml, about 17mg/ml, about 16 mg/ml, about 15 mg/ml, about 14 mg/ml, about 13 mg/ml,about 12 mg/ml, about 11 mg/ml, about 10 mg/ml, about 9 mg/ml, about 8mg/ml, about 7 mg/ml, about 6 mg/ml, about 5 mg/ml, about 4 mg/ml, about3 mg/ml, about 2 mg/ml, about 1 mg/ml, or about 0.1 mg/ml. In someembodiments, the compositions may contain an analog at a concentrationrange of A to B mg/ml, for example, about 0.001 to about 30.0 mg/ml.

Additional dosing guidance can be gauged from other antibodytherapeutics, such as bevacizumab (Avastin™ Genentech); Cetuximab(Exbitux™ Imclone), Panitumumab (Vectibix™ Amgen), and Trastuzumab(Herceptin™ Genentech).

Timing of Administration

The disclosed pharmaceutical formulations may be administered accordingto any regimen including, for example, daily (1 time per day, 2 timesper day, 3 times per day, 4 times per day, 5 times per day, 6 times perday), every two days, every three days, every four days, every fivedays, every six days, weekly, bi-weekly, every three weeks, monthly, orbi-monthly. Timing, like dosing can be fine-tuned based on dose-responsestudies, efficacy, and toxicity data, and initially gauged based ontiming used for other antibody therapeutics.

Controlled Release Formulations

The pharmaceutical composition is in certain aspects modified into adepot form, such that the manner in which the active ingredients of thepharmaceutical composition (e.g., the binding agents) is released intothe body to which it is administered is controlled with respect to timeand location within the body (see, for example, U.S. Pat. No.4,450,150). Depot forms in various aspects, include, for example, animplantable composition comprising a porous or non-porous material, suchas a polymer, wherein the binding agents are encapsulated by or diffusedthroughout the material and/or degradation of the non-porous material.The depot is then implanted into the desired location within the bodyand the binding agents are released from the implant at a predeterminedrate.

Accordingly, the pharmaceutical composition in certain aspects ismodified to have any type of in vivo release profile. In some aspects,the pharmaceutical composition is an immediate release, controlledrelease, sustained release, extended release, delayed release, orbi-phasic release formulation. Methods of formulating peptides (e.g.,peptide binding agents) for controlled release are known in the art.See, for example, Qian et al., J Pharm 374: 46-52 (2009) andInternational Patent Application Publication Nos. WO 2008/130158,WO2004/033036; WO2000/032218; and WO 1999/040942. Suitable examples ofsustained-release preparations include semipermeable polymer matrices inthe form of shaped articles, e.g., films, or microcapsules. Sustainedrelease matrices include polyesters, hydrogels, polylactides (U.S. Pat.No. 3,773,919, EP 58,481), copolymers of L-glutamic acid and gammaethyl-L-glutamate (Sidman, et al., Biopolymers, 22: 547-556 (1983)),poly (2-hydroxyethyl-methacrylate) (Langer, et al., J. Biomed. Mater.Res., 15:167-277 (1981) and Langer, Chem. Tech., 12:98-105 (1982)),ethylene vinyl acetate (Langer, et al, supra) orpoly-D(−)-3-hydroxybutyric acid (EP 133,988). Sustained-releasecompositions also may include liposomes, which can be prepared by any ofseveral methods known in the art (e.g., DE 3,218,121; Epstein, et al.,Proc. Natl. Acad. Sci. USA, 82:3688-3692 (1985); Hwang, et al., Proc.Natl. Acad. Sci. USA, 77:4030-4034 (1980); EP 52,322; EP 36,676; EP88,046; EP 143,949).

Combinations

The compositions of the disclosures may be employed alone, or incombination with other agents. In some embodiments, more than one typeof binding agent are administered. For example, the administeredcomposition, e.g., pharmaceutical composition, may comprise an antibodyas well as an scFv. In some embodiments, the compositions of thedisclosure are administered together with another therapeutic agent ordiagnostic agent, including any of those described herein. Certaindiseases, e.g., cancers, or patients may lend themselves to a treatmentof combined agents to achieve an additive or even a synergistic effectcompared to the use of any one therapy alone.

Uses

Based in part on the data provided herein, the binding agents,conjugates, host cells, populations of cells, and pharmaceuticalcompositions are useful for treating a neoplasm, tumor, or a cancer.

For purposes of the present disclosure, the term “treat” and “prevent”as well as words stemming therefrom, as used herein, do not necessarilyimply 100% or complete treatment (e.g., cure) or prevention. Rather,there are varying degrees of treatment or prevention of which one ofordinary skill hi the art recognizes as having a potential benefit ortherapeutic effect. In this respect, the methods of the presentdisclosures can provide any amount or any level of treatment orprevention of a cancer in a patient, e.g., a human. Furthermore, thetreatment or prevention provided by the method disclosed herein caninclude treatment or prevention of one or more conditions or symptoms ofthe disease, e.g., cancer, being treated or prevented. Also, forpurposes herein, “prevention” can encompass delaying the onset of thedisease, or a symptom or condition thereof.

The materials and methods described herein are especially useful forinhibiting neoplastic cell growth or spread; particularly neoplasticcell growth for which the IL13Rα2 targeted by the binding agents plays arole.

Neoplasms treatable by the binding agents, conjugates, host cells,populations of cells, and pharmaceutical compositions of the disclosuresinclude solid tumors, for example, carcinomas and sarcomas. Carcinomasinclude malignant neoplasms derived from epithelial cells whichinfiltrate, for example, invade, surrounding tissues and give rise tometastases. Adenocarcinomas are carcinomas derived from glandulartissue, or from tissues that form recognizable glandular structures.Another broad category of cancers includes sarcomas and fibrosarcomas,which are tumors whose cells are embedded in a fibrillar or homogeneoussubstance, such as embryonic connective tissue. The invention alsoprovides methods of treatment of cancers of myeloid or lymphoid systems,including leukemias, lymphomas, and other cancers that typically are notpresent as a tumor mass, but are distributed in the vascular orlymphoreticular systems. Further contemplated are methods for treatmentof adult and pediatric oncology, growth of solid tumors/malignancies,myxoid and round cell carcinoma, locally advanced tumors, cancermetastases, including lymphatic metastases. The cancers listed hereinare not intended to be limiting. Both age (child and adult), sex (maleand female), primary and secondary, pre- and post-metastatic, acute andchronic, benign and malignant, anatomical location cancer embodimentsand variations are contemplated targets. Cancers are grouped byembryonic origin (e.g., carcinoma, lymphomas, and sarcomas), by organ orphysiological system, and by miscellaneous grouping. Particular cancersmay overlap in their classification, and their listing in one group doesnot exclude them from another.

Carcinomas that may be targeted include adrenocortical, acinar, aciniccell, acinous, adenocystic, adenoid cystic, adenoid squamous cell,cancer adenomatosum, adenosquamous, adnexel, cancer of adrenal cortex,adrenocortical, aldosterone-producing, aldosterone-secreting, alveolar,alveolar cell, ameloblastic, ampullary, anaplastic cancer of thyroidgland, apocrine, basal cell, basal cell, alveolar, comedo basal cell,cystic basal cell, morphea-like basal cell, multicentric basal cell,nodulo-ulcerative basal cell, pigmented basal cell, sclerosing basalcell, superficial basal cell, basaloid, basosquamous cell, bile duct,extrahepatic bile duct, intrahepatic bile duct, bronchioalveolar,bronchiolar, bronchioloalveolar, bronchoalveolar, bronchoalveolar cell,bronchogenic, cerebriform, cholangiocellular, chorionic, choroidsplexus, clear cell, cloacogenic anal, colloid, comedo, corpus, cancer ofcorpus uteri, cortisol-producing, cribriform, cylindrical, cylindricalcell, duct, ductal, ductal cancer of the prostate, ductal cancer in situ(DCIS), eccrine, embryonal, cancer en cuirasse, endometrial, cancer ofendometrium, endometroid, epidermoid, cancer ex mixed tumor, cancer expleomorphic adenoma, exophytic, fibrolamellar, cancer fibrosum,follicular cancer of thyroid gland, gastric, gelatinform, gelatinous,giant cell, giant cell cancer of thyroid gland, cancer gigantocellulare,glandular, granulose cell, hepatocellular, Hurthle cell, hypernephroid,infantile embryonal, islet cell carcinoma, inflammatory cancer of thebreast, cancer in situ, intraductal, intraepidermal, intraepithelial,juvenile embryonal, Kulchitsky-cell, large cell, leptomeningeal,lobular, infiltrating lobular, invasive lobular, lobular cancer in situ(LCIS), lymphoepithelial, cancer medullare, medullary, medullary cancerof thyroid gland, medullary thyroid, melanotic, meningeal, Merkel cell,metatypical cell, micropapillary, cancer molle, mucinous, cancermuciparum, cancer mucocellulare, mucoepidermoid, cancer mucosum, mucous,nasopharyngeal, neuroendocrine cancer of the skin, noninfiltrating,non-small cell, non-small cell lung cancer (NSCLC), oat cell, cancerossificans, osteoid, Paget's disease of the bone or breast, papillary,papillary cancer of thyroid gland, periampullary, preinvasive, pricklecell, primary intrasseous, renal cell, scar, schistosomal bladder,Schneiderian, scirrhous, sebaceous, signet-ring cell, cancer simplex,small cell, small cell lung cancer (SCLC), spindle cell, cancerspongiosum, squamous, squamous cell, terminal duct, anaplastic thyroid,follicular thyroid, medullary thyroid, papillary thyroid, trabecularcancer of the skin, transitional cell, tubular, undifferentiated cancerof thyroid gland, uterine corpus, verrucous, villous, cancer villosum,yolk sac, squamous cell particularly of the head and neck, esophagealsquamous cell, and oral cancers and carcinomas.

Sarcomas that may be targeted include adipose, alveolar soft part,ameloblastic, avian, botryoid, sarcoma botryoides, chicken,chloromatous, chondroblastic, clear cell sarcoma of kidney, embryonal,endometrial stromal, epithelioid, Ewing's, fascial, fibroblastic, fowl,giant cell, granulocytic, hemangioendothelial, Hodgkin's, idiopathicmultiple pigmented hemorrhagic, immunoblastic sarcoma of B cells,immunoblastic sarcoma of T cells, Jensen's, Kaposi's, Kupffer cell,leukocytic, lymphatic, melanotic, mixed cell, multiple, lymphangio,idiopathic hemorrhagic, multipotential primary sarcoma of bone,osteoblastic, osteogenic, parosteal, polymorphous, pseudo-Kaposi,reticulum cell, reticulum cell sarcoma of the brain, rhabdomyosarcoma,Rous, soft tissue, spindle cell, synovial, telangiectatic, sarcoma(osteosarcoma)/malignant fibrous histiocytoma of bone, and soft tissuesarcomas.

Lymphomas that may targeted include AIDS-related, non-Hodgkin's,Hodgkin's, T-cell, T-cell leukemia/lymphoma, African, B-cell, B-cellmonocytoid, bovine malignant, Burkitt's, centrocytic, lymphoma cutis,diffuse, diffuse, large cell, diffuse, mixed small and large cell,diffuse, small cleaved cell, follicular, follicular center cell,follicular, mixed small cleaved and large cell, follicular,predominantly large cell, follicular, predominantly small cleaved cell,giant follicle, giant follicular, granulomatous, histiocytic, largecell, immunoblastic, large cleaved cell, large noncleaved cell,Lennert's, lymphoblastic, lymphocytic, intermediate; lymphocytic,intermediately differentiated, plasmacytoid; poorly differentiatedlymphocytic, small lymphocytic, well differentiated lymphocytic,lymphoma of cattle; MALT, mantle cell, mantle zone, marginal zone,Mediterranean lymphoma mixed lymphocytic-histiocytic, nodular,plasmacytoid, pleomorphic, primary central nervous system, primaryeffusion, small b-cell, small cleaved cell, small noncleaved cell,T-cell lymphomas; convoluted T-cell, cutaneous t-cell, small lymphocyticT-cell, undefined lymphoma, u-cell, undifferentiated, aids-related,central nervous system, cutaneous T-cell, effusion (body cavity-based),thymic lymphoma, and cutaneous T cell lymphomas.

Leukemias and other blood cell malignancies that may be targeted includeacute lymphoblastic, acute myeloid, lymphocytic, chronic myelogenous,hairy cell, lymphoblastic, myeloid, lymphocytic, myelogenous, leukemia,hairy cell, T-cell, monocytic, myeloblastic, granulocytic, gross, handmirror-cell, basophilic, hemoblastic, histiocytic, leukopenic,lymphatic, Schilling's, stem cell, myelomonocytic, prolymphocytic,micromyeloblastic, megakaryoblastic, megakaryocytic, Rieder cell,bovine, aleukemic, mast cell, myelocytic, plasma cell, subleukemic,multiple myeloma, nonlymphocytic, and chronic myelocytic leukemias.

Brain and central nervous system (CNS) cancers and tumors that may betargeted include astrocytomas (including cerebellar and cerebral),gliomas (including malignant gliomas, glioblastomas, brain stem gliomas,visual pathway and hypothalamic gliomas), brain tumors, ependymoma,medulloblastoma, supratentorial primitive neuroectodermal tumors,primary central nervous system lymphoma, extracranial germ cell tumor,myelodysplastic syndromes, oligodendroglioma,myelodysplastic/myeloproliferative diseases, myelogenous leukemia,myeloid leukemia, multiple myeloma, myeloproliferative disorders,neuroblastoma, plasma cell neoplasm/multiple myeloma, central nervoussystem lymphoma, intrinsic brain tumors, astrocytic brain tumors, andmetastatic tumor cell invasion in the central nervous system.

Gastrointestinal cancers that may be targeted include extrahepatic bileduct cancer, colon cancer, colon and rectum cancer, colorectal cancer,gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoidtumor, gastrointestinal carcinoid tumors, gastrointestinal stromaltumors, bladder cancers, islet cell carcinoma (endocrine pancreas),pancreatic cancer, islet cell pancreatic cancer, prostate cancer rectalcancer, salivary gland cancer, small intestine cancer, colon cancer, andpolyps associated with colorectal neoplasia. A discussion of colorectalcancer is described in Barderas et al., Cancer Research 72: 2780-2790(2012).

Bone cancers that may be targeted include osteosarcoma and malignantfibrous histiocytomas, bone marrow cancers, bone metastases,osteosarcoma/malignant fibrous histiocytoma of bone, and osteomas andosteosarcomas. Breast cancers that may be targeted include small cellcarcinoma and ductal carcinoma.

Lung and respiratory cancers that may be targeted include bronchialadenomas/carcinoids, esophagus cancer esophageal cancer, esophagealcancer, hypopharyngeal cancer, laryngeal cancer, hypopharyngeal cancer,lung carcinoid tumor, non-small cell lung cancer, small cell lungcancer, small cell carcinoma of the lungs, mesothelioma, nasal cavityand paranasal sinus cancer, nasopharyngeal cancer, nasopharyngealcancer, oral cancer, oral cavity and lip cancer, oropharyngeal cancer;paranasal sinus and nasal cavity cancer, and pleuropulmonary blastoma.

Urinary tract and reproductive cancers that may be targeted includecervical cancer, endometrial cancer, ovarian epithelial cancer,extragonadal germ cell tumor, extracranial germ cell tumor, extragonadalgerm cell tumor, ovarian germ cell tumor, gestational trophoblastictumor, spleen, kidney cancer, ovarian cancer, ovarian epithelial cancer,ovarian germ cell tumor, ovarian low malignant potential tumor, penilecancer, renal cell cancer (including carcinomas), renal cell cancer,renal pelvis and ureter (transitional cell cancer), transitional cellcancer of the renal pelvis, and ureter, gestational trophoblastic tumor,testicular cancer, ureter and renal pelvis, transitional cell cancer,urethral cancer, endometrial uterine cancer, uterine sarcoma, vaginalcancer, vulvar cancer, ovarian carcinoma, primary peritoneal epithelialneoplasms, cervical carcinoma, uterine cancer and solid tumors in theovarian follicle), superficial bladder tumors, invasive transitionalcell carcinoma of the bladder, and muscle-invasive bladder cancer.

Skin cancers and melanomas (as well as non-melanomas) that may betargeted include cutaneous t-cell lymphoma, intraocular melanoma, tumorprogression of human skin keratinocytes, basal cell carcinoma, andsquamous cell cancer. Liver cancers that may be targeted includeextrahepatic bile duct cancer, and hepatocellular cancers. Eye cancersthat may be targeted include intraocular melanoma, retinoblastoma, andintraocular melanoma Hormonal cancers that may be targeted include:parathyroid cancer, pineal and supratentorial primitive neuroectodermaltumors, pituitary tumor, thymoma and thymic carcinoma, thymoma, thymuscancer, thyroid cancer, cancer of the adrenal cortex, and ACTH-producingtumors.

Miscellaneous other cancers that may be targeted include advancedcancers, AIDS-related, anal cancer, adrenal, cortical, aplastic anemia,aniline, betel or buyo cheek, cerebriform, chimney-sweeps, clay pipe,colloid, contact, cystic, dendritic, cancer a deux, duct, dye workers,encephaloid, cancer en cuirasse, endometrial, endothelial, epithelial,glandular, cancer in situ, kang, kangri, latent, medullary, melanotic,mule-spinners', non-small cell lung, occult cancer, paraffin, pitchworkers', scar, schistosomal bladder, scirrhous, lymph node, small celllung, soft, soot, spindle cell, swamp, tar, and tubular cancers.

Miscellaneous other cancers that may be targeted also include carcinoid(gastrointestinal and bronchial) Castleman's disease chronicmyeloproliferative disorders, clear cell sarcoma of tendon sheaths,Ewing's family of tumors, head and neck cancer, lip and oral cavitycancer, Waldenstrom's macroglobulinemia, metastatic squamous neck cancerwith occult primary, multiple endocrine neoplasia syndrome, multiplemyeloma/plasma cell neoplasm, Wilms' tumor, mycosis fungoides,pheochromocytoma, sezary syndrome, supratentorial primitiveneuroectodermal tumors, unknown primary site, peritoneal effusion,malignant pleural effusion, trophoblastic neo-plasms, andhemangiopericytoma.

In exemplary aspects, the cancer is any one of the foregoing describedin which IL13Rα2 is expressed on the cells of the cancer. In exemplaryaspects, the cancer is colon cancer. In exemplary aspects, the cancer isGlioblastoma Multiforme. In exemplary aspects, the method of treatingcancer in a subject in need thereof comprises administering to thesubject any of the binding agents, conjugates, nucleic acids, vectors,host cells, cell populations, or pharmaceutical compositions describedherein, in an amount effective to treat the cancer. In exemplaryaspects, the method comprises administering a conjugate describedherein. In exemplary aspects, the method comprises administering hostcells of the disclosures and the host cells are autologous cells inrelation to the subject being treated. In exemplary aspects, the methodcomprises administering host cells of the disclosures and the host cellsare cells obtained from the subject being treated. In exemplary aspects,the cells are T-lymphocytes. In alternative aspects, the cells arenatural killer cells.

The disclosure will be more fully understood by reference to thefollowing examples, which detail exemplary embodiments of thedisclosure. The examples should not, however, be construed as limitingthe scope of the disclosure.

Example 1

Materials

Lipofectamine 2000 and the pEF6/Myc-His vector were obtained fromInvitrogen. Monoclonal antibodies to IL13Rα2 (clones YY-23Z and B-D13)and the IsoStrip mouse monoclonal antibody isotyping kit were purchasedfrom Santa Cruz Biotechnology (Santa Cruz, Calif.). The mAb to IL13Rα2(clone 83807) and recombinant human and mouse IL13Rα2hFc and IL13Rα1hFcchimeras were purchased from R&D Systems (Minneapolis, Minn.).Biotinylated horse anti-mouse antibodies and the Elite kit were obtainedfrom Vector Laboratories (Burlingame, Calif.). 3,3′-Diaminobenzidinesubstrate was purchased from Dako (Carpinteria, Calif.). Goat anti-mouseantibody conjugated with peroxidase was purchased from ChemiconInternational (Temicula, Calif.), and Pngase F was purchased from NewEngland Biolabs (Ipswich, Mass.). The QuikChange Lightning™site-directed mutagenesis kit was purchased from Agilent Technologies,Inc. (Santa Clara, Calif.), and the RNeasy Plus™ kit was received fromQiagen (Valencia, Calif.). The cDNA iScript™ kit, 7.5% Tris-HCl gel, andImmunStar™ WesternC™ developing reagent and protein marker werepurchased from Bio-Rad. The human IL-13 ELISA kit was purchased fromeBioscience (San Diego, Calif.). GBM12 and GBM43 were kindly provided byDr. David C. James (University of California-San Francisco), and thecDNA encoding human wild-type IL13Rα2 was obtained from Dr. WaldemarDebinski (Wake Forest University). Obtaining the cDNA encoding the humanwild-type IL13Rα2 or most other proteins involves the use of well-knowntechniques and readily available reagents.

Cell Lines

U373 (GBM), 293T (human embryonic kidney), and Raji (Burkitt's lymphoma)cell line were purchased from the American Type Culture Collection(ATCC; Manassas, Va.). The generation of U373 cells expressing enhancedgreen fluorescent protein and firefly luciferase (U373.eGFP.ffLuc), 293Tcells expressing green fluorescent protein (293T.GFP) or IL13Rα2 and GFP(293T.IL13Rα2.GFP) were previously reported. See Chow et al., Mol. Ther.21:629-637 (2013); Krebs et al., Cytotherapy 16:1121-1131 (2014). Celllines were grown in RPMI or DMEM (Thermo Scientific HyClone, Waltham,Mass.; Lonza, Basel, Switzerland) with 10% fetal calf serum (FCS;HyClone, Logan, Utah) and 2 mM GlutaMAX-I™ (Invitrogen, Carlsbad,Calif.). The Characterized Cell Line Core Facility at MD Anderson CancerCenter, Houston, Tex., performed cell line validation.

Immunization

To obtain monoclonal antibodies with specificity to native IL13Rα2, thehuman recombinant IL13Rα2hFc fusion was used for immunization of animalsand in all screening assays. Two 6-week-old female BALB/c mice wereimmunized with intraperitoneal injection of 10 μg of rhIL13Rα2hFcprotein in complete Freund's adjuvant followed by intraperitonealinjection of 10 μg of rhIL13Rα2hFc protein in incomplete Freund'sadjuvant at a 2-week interval for 2 months. Two weeks after the lastintraperitoneal injection and 3 days before the fusion, a boost wasperformed by the combination of intravenous and intraperitonealinjection of 10 μg of antigen without Freund's adjuvant. The fusion ofmouse spleen cells with the mouse myeloma cell line X63.Ag8.653 subcloneP3O1 was performed by using a procedure described by Köhler and Milstein(27). Hybridoma supernatants were assayed for the presence of IL13Rα2antibodies using an enzyme-linked immunosorbent assay (ELISA). Selectedpopulations were cloned, and supernatants were assayed to identify theclones with strongest binding.

Generation of CHO Cell Line Expressing Human IL13Rα2

The cDNA encoding human wild-type IL13Rα2 was amplified with thefollowing primer pair: forward, 5′-GCTTGGTACCGAATGGCTTTCGTTTGCTTGGC-3′(SEQ ID NO: 17) and reverse, 5′-GTTTTTGTTCGAATGTATCACAGAAAAATTCTGG-3′(SEQ ID NO: 18). The purified PCR product was restricted with KpnI andBstBI enzymes, agarose gel-purified, and subsequently cloned into thepEF6/Myc-His vector in a reading frame with Myc and His6 tags. CHO cellswere plated at 80% confluence and transfected with a plasmid encodingthe IL13Rα2 using Lipofectamine 2000. The following day, 4 μg/mlblasticidin was added for selection of cells that had stablyincorporated and expressed the IL13Rα2 transcript. A stable populationof cells was further subcloned in 96-well plates at a density of onecell/well. Ten days later, single clones were screened by flow cytometryfor cell surface expression of IL13Rα2 using an antibody to IL13Rα2(clone B-D13). The clone with the highest level of IL13Rα2 expressionwas selected and expanded for subsequent screening of hybridomassecreting IL13Rα2 antibodies.

ELISA

96-well plates were coated with 50 μl of human or mouse recombinantIL13Rα2hFc or IL13Rα1hFc or human control IgG at a concentration of 1μg/ml overnight at 4° C. Following washes with TBS-Tween 20 buffer andblocking with 1% nonfat dry milk, 50 μl of purified antibodies, serum,or hybridoma supernatants at various dilutions were applied to the plateand incubated for 1 hour at room temperature. Bound antibodies weredetected with goat anti-mouse antibodies conjugated to alkalinephosphatase following the development with alkaline phosphatasesubstrate. Plates were read at A405 using a UniRead 800 plate reader(BioTek).

Flow Cytometry

CHO or HEK cells expressing IL13Rα2; the glioma cell lines A172, N10,U251, U87, and U118; patient-derived GBM12 and GBM43, and primary humanastrocytes were stained with IL13Rα2 (clone 47) monoclonal antibody at 1μg/ml followed by goat anti-mouse Alexa Fluor 647 (1:500). All stainingprocedures were performed on ice. Samples were analyzed using the BDFACSCanto flow cytometer and FACSDiVa™ software.

For the experiments disclosed in Examples 13-16, a FACSCaliburinstrument (BD Bioscience, Mountain View, Calif.) was used to acquireimmunofluorescence data that were analyzed with CellQuest (BD) or FCSExpress software (De Novo Software, Los Angeles, Calif.). Isotypecontrols were immunoglobulin G1-fluorescein isothiocyanate (IgG1-FITC;BD Bioscience) and IgG1-phycoerythrin (IgG1-PE; BD Bioscience). SSR47-CAR expression was detected by staining T cells with an IL13Rα2chimera followed by Fc-FITC (Milipore) or Fc-PE (SouthernBiotech). LSR47-CARs were detected using Fc-FITC or Fc-PE. U373 cells were analyzedfor PD-L1 expression using a CD271 PE antibody (BD Bioscience). Forward-and side-scatter gating were used to discriminate live cells from deadcells. Cells were collected and washed once with phosphate-bufferedsaline (PBS) containing 1% FBS (Sigma; FACS buffer) prior to theaddition of antibodies. Cell were incubated for 30 minutes on ice in thedark, washed once, and fixed in 0.5% paraformaldehyde/FACS buffer priorto analysis.

PCR

To determine the expression of IL13Rα2 in various glioma cells andastrocytes, total RNA was generated from the cell pellets using theRNeasy Plus kit. 200 ng of total RNA was then converted into cDNA usingthe cDNA iScript kit. The cDNA was further amplified by PCR for IL13Rα2and GAPDH for 30 cycles using IL13Rα2 and GAPDH primers and visualizedon a 1% agarose gel.

Surface Plasmon Resonance

The affinity and rates of interaction between IL13Rα2 (clone 47)monoclonal antibody, commercially available IL13Rα2 monoclonalantibodies (clones 83807 and B-D13), and target (rhIL13Rα2) weremeasured with a Biacore 3000 biosensor through surface plasmon resonance(SPR). The monoclonal antibodies were immobilized (covalently) to thedextran matrix of the sensor chip (CMS) using the amino coupling kit.The carboxyl groups on the sensor surfaces were activated with aninjection of a solution containing 0.2MN-ethyl-N′-(3-diethylamino-propyl)-carbodiimide and 0.05MN-hydroxysuccinimide. The immobilization procedure was completed by theinjection of 1Methanolamine hydrochloride to block the remaining estergroups. All steps of the immobilization process were carried out at aflow rate of 10 μl/minute. The control surface was prepared similarlywith the exception that running buffer was injected rather thanmonoclonal antibodies. Binding reactions were performed at 25° C. inHBS-P buffer (20 mM HEPES, pH 7.4, 150 mM NaCl, and 0.005% (v/v)surfactant P20) using a flow rate of 20 μl/minute. Target (rhIL13Rα2)was added at various concentrations in the flow during the bindingphase. The amount of protein bound to the sensor chip was monitored bythe change in refractive index (represented by response units (RU)). Theinstrument was programmed to perform a series of binding measurementswith increasing concentrations of target over the same surface.Triplicate injections of each concentration of target were performed.Sensorgrams (plots of changes in RU on the surface as a function oftime) were analyzed using BIAevaluation v4.1. Affinity constants wereestimated by curve fitting using a 1:1 binding model.

Data Preparation and Kinetic Analysis

The estimation of kinetic parameters was performed by repetitiveinjections of a range of target concentrations over the immobilizedmAbs. Data were prepared by the method of “double referencing.” Thismethod utilizes parallel injections of each target sample over a controldextran surface as well as running buffer injections over both theimmobilized mAbs and control dextran surfaces. Subtraction of thesesensorgrams yielded the control; this was subtracted from theexperimental sensorgram. Each data set (consisting of sensorgrams ofincreasing target concentrations over the same level of immobilizedmAbs) was analyzed using various kinetic models. The BIAevaluation v 4.1software was then used for data analysis. Affinity constants wereestimated by curve fitting using a 1:1 binding model. Sensorgramassociation and dissociation curves were fit locally or globally. Therate of complex formation during the sample injection is described by anequation of the following type: dR/dt=k_(a)C (R_(max)−R)−k_(d)R (for a1:1 interaction) where R is the SPR signal in RU, C is the concentrationof analyte, R_(max) is the maximum analyte binding capacity in RU, anddR/dt is the rate of change of SPR signal. The early binding phase (300s) was used to determine the association constant (k_(a)) between mAband target. The dissociation phase (k_(d)) was measured using the rateof decline in RU on introduction of free buffer at the end of targetinjections. Data were simultaneously fit by the software program (globalfitting algorithm), and the dissociation constant (K_(D)) of thecomplexes was determined as the ratio k_(a)/k_(d). For quantitativeanalysis, three independent replicates were performed for each sample.Data are expressed as mean±S.E.

Competitive Binding Assay

For the competitive binding plate assay, a 96-well plate was coated with50 μl of affinity-purified hrIL13Rα2hFc at 1 μg/ml in carbonate buffer,pH 9.6 and stored overnight at 4° C. After washing with PBS containing0.05% Tween 20, mAbs to IL13Rα2 (10 μg/ml) or control mIgG were addedfor 30 minutes at room temperature. After washing, 50 μl of purifiedrhIL-13 in PBS and 0.1% BSA at 10 ng/ml were added for a 1-hourincubation at room temperature and assayed for bound rhIL-13 usingdetection reagents from a human IL-13 ELISA kit. Separately, HEK cellsexpressing wild-type IL13Rα2 or 4-amino-acid mutants (see Example 10) inthe IL13Rα2 sequence were pretreated with either rhIL-13 or mAb IL13Rα2(clone 47) at 2 μg/ml for 30 minutes on ice followed by a 1-hourincubation with IL13Rα2 (clone 47) mAb or rhIL-13 at 100 ng/ml,respectively. Binding of rhIL-13 to IL13Rα2 alone or in the presence ofcompetitor was detected with human IL-13 mAb-FITC. Binding of IL13Rα2(clone 47) mAb to rhIL13Rα2 alone or in the presence of competitor wasdetected with anti-mouse antibody conjugated to Alexa Fluor 649 andanalyzed by flow cytometry.

Mutagenesis of IL13Rα

Previously, Tyr²⁰⁷, Asp²⁷¹, Tyr³¹⁵, and Asp³¹⁸ of the human IL13Rα2 wereidentified as residues crucial for interaction with human IL-13 (28). Todetermine whether those residues were important for binding of IL13Rα2(clone 47) mAb to IL13Rα2, the Tyr²⁰⁷, Asp²⁷¹, Tyr³¹⁵, and Asp³¹⁸residues were mutated to Ala separately or at the same time(4-amino-acid mutant) using the QuikChange Lightning site-directedmutagenesis kit according to the manufacturer's recommendations.Sequencing of selected clones was performed using conventionaltechniques, which confirmed the presence of the selected mutation. HEKcells were transfected with wild-type or mutated variants of IL13Rα2cDNA in the pEF6 Myc-His vector using Lipofectamine Plus transfectionreagent. 48 hours after transfection, the cells were collected andanalyzed for binding to IL13Rα2 (clone 47) mAb via flow cytometry.

Western Blot

The rhIL13Rα2 was applied to a 7.5% Tris-HCl gel (Bio-Rad) at 200ng/lane and resolved under reducing conditions. After the transfer ofproteins to a PVDF membrane (Bio-Rad) and blocking with 2% nonfat drymilk, the membrane was stained with anti-IL13Rα2 mAb (clones YY-23Z andB-D13) at 2 μg/ml or with supernatant collected from hybridoma clones(diluted 10 times), followed by goat anti-mouse antibody conjugated toperoxidase. ImmunStar™ WesternC™ was used to develop reactions. Imageswere captured using a Bio-Rad ChemiDoc imaging system.

For experiments disclosed in Examples 13-16, cells were dissociated withPBS+3 mM EDTA and lysed in a buffer containing 50 mM Tris, 150 mM NaCl,5 mM EDTA, 1% Triton X-100 (all from Sigma, St. Louis, Mo.), andprotease inhibitors (Thermo Scientific, Waltham, Mass.). Proteinconcentrations were determined using a Bio-Rad protein assay (Bio-Rad,Hercules, Calif.) with bovine serum albumin (BSA) as the standard.Samples were denatured in Laemmli buffer (Bio-Rad) at 95° C. for 5minutes. 5 μg of protein were loaded per well and run on a 10%polyacrylamide gel. Proteins were transferred to nitrocellulosemembranes (BioRad). Membranes were blocked with 5% milk powder (MP) inTris-buffered saline (TBS)+0.1% Tween-20 (Sigma) and then probed withanti-CD3.ζ (sc-1239, Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.)or GAPDH (sc-47724, Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.)mouse monoclonal antibodies followed by a horseradish peroxidase (HRP)conjugated goat anti-mouse IgG antibody (sc-2005, Santa CruzBiotechnology, Inc.). Blots were developed using SuperSignal West DuraExtended Duration Substrate (Thermo Scientific) and exposed to GeneMateBlue Basic Autoradiography Film (BioExpress, Kaysville, Utah).

Immunohistochemistry

The GBM tissues were collected in accordance with a protocol approved bythe Institutional Review Board at the University of Chicago.Flash-frozen brain-tumor tissues were cut to a thickness of 10 μm.Tissue sections were fixed with −20° C. methanol and stained for humanIL13Rα2 using mouse IL13Rα2 (clone 47) mAb at a concentration of 3 μg/mlor isotype control mIgG1. The bound antibodies were detected withbiotinylated horse anti-mouse antibodies (1:100). The antigen-antibodybinding was detected by the Elite kit with 3,3′-diaminobenzidinesubstrate. Slides were analyzed using the CRI Panoramic Scan Whole SlideScanner and Panoramic Viewer software.

Animal Study

All animals were maintained and cared for in accordance with theInstitutional Animal Care and Use Committee protocol and according toNational Institutes of Health guidelines. The animals used in theexperiments were 6- to 7-week-old male athymic nu/nu mice. Mice wereanesthetized with an intraperitoneal injection of ketaminehydrochloride/xylazine (25 mg/ml/2.5 mg/ml) mixture. To establishintracranial tumors, a midline cranial incision was made, and aright-sided burr hole was placed 2 mm lateral to the sagittal suture andabout 2 mm superior to λ. Animals were positioned in a stereotacticframe, and a Hamilton needle was inserted through the burr hole andadvanced 3 mm. Intracranial penetration was followed by (i) injection of2.5×10⁴ U251 glioma cells in 2.5 μl of sterile PBS in combination with200 ng of mIgG or IL13Rα2 (clone 47) mAb or (ii) 3 dayspost-intracranial injection of glioma cells with PBS or 10 μg of IL13Rα2(clone 47 or B-D13) mAb as described previously (29, incorporated hereinby reference). All mice were monitored for survival. Three animals fromeach group were sacrificed at day 17, and brains were harvested andfrozen for sectioning, hematoxylin and eosin (H&E) staining, andmicroscopic analysis.

Animal experiments disclosed in Examples 13-16 followed a protocolapproved by the Baylor College of Medicine Institutional Animal Care andUse Committee. Experiments were performed as described in Ahmed et al.,Clin. Cancer Res. 16:474-485 (2010) (incorporated herein by reference)with a few modifications. ICR-SCID mice were purchased from Taconic(IcrTac:ICR-Prkdcscid; Fox Chase C.B-17 SCID™ ICR; Taconic, Hudson,N.Y.). Male 7- to 9-week-old mice were anesthetized, head were shavedand the mice were immobilized in a Cunningham™ Mouse/Neonatal RatAdaptor (Stoelting, Wood Dale, Ill.) stereotactic apparatus fitted intoan E15600 Lab Standard Stereotaxic Instrument (Stoelting), and thenscrubbed with 1% povidone-iodine. A 10 mm skin incision was made alongthe midline. The tip of a 30G ½ inch needle mounted on a Hamiltonsyringe (Hamilton, Reno, Nev.) served as the reference point. A 1 mmburr-hole was drilled into the skull 1 mm anterior and 2 mm to the rightof the bregma. 1×10⁵ U373.eGFP.ffLuc cells in 2.0 μL were injected 3 mmdeep to the bregma, corresponding to the center of the right caudatenucleus over 5 minutes. The needle was left in place for 3 minutes toavoid tumor cell extrusion, and then withdrawn over 5 minutes. Sevendays after tumor cell injection, animals were treated with 2×10⁶effector cells in 2 μL to the same tumor coordinates. The incision wasclosed with 2-3 interrupted 7.0 Ethilon sutures (Ethicon, Inc.,Somerville, N.J.). A subcutaneous injection of 0.03-0.1 mg/kgbuprenorphine (Buprenex® RBH, Hull, England) was given for pain control.

Generation of Retroviral Vectors Encoding IL13Rα2-scFv-Specific CARs

A codon-optimized gene was synthesized by GeneArt (Invitrogen, Carlsbad,Calif.) containing the immunoglobulin heavy-chain leader peptide37, andscFv47 flanked by 5′ NcoI and 3′ BamHI sites. This mini gene wassubcloned into SFG retroviral vector containing IL13Rα2-specific CARs(47-CARs) with short or long spacer regions (SSRs, LSRs) and CD28.ζ,CD28.OX40.ζ, CD28.41BB.ζ, or 41BB.ζ endodomains.5,38,39 All CARscontained a CD28 transmembrane domain except for 47.SSR.CAR.41BB.ζ,which had a CD8α transmembrane domain. 47.SSR.CAR and 47.LSR.CAR withoutan endodomain (47.SSR.CAR.Δ and 47.LSR.CAR.Δ) were generated by PCRcloning. All cloning of the CARs were verified by sequencing (Seqwright,Houston, Tex.). RD114-pseudotyped retroviral particles were generated bytransient transfection of 293T cells as previously described in Johnsonet al., Sci. Transl. Med. 7:275ra22 (2015), incorporated herein byreference.

Generation of CAR T Cells

Human peripheral blood mononuclear cells (PBMCs) from healthy donorswere obtained under a Baylor College of Medicine IRB-approved protocol,after informed consent was obtained in accordance with the Declarationof Helsinki. To generate 47-CAR T cells, PBMCs were isolated byLymphoprep (Greiner Bio-One, Monroe, N.C.) gradient centrifugation andthen stimulated on non-tissue culture treated 24-well plates, which wereprecoated with OKT3 (CRL-8001, ATCC) and CD28 (BD Bioscience, MountainView, Calif.) antibodies. Recombinant human interleukin-7 (IL7) and IL15(IL7, 10 ng/mL; IL15, 5 ng/mL; Proleukin; Chiron, Emeryville, Calif.)were added to cultures on day 2 (Xu et al., Blood 123:3750-3759 (2014),incorporated herein by reference). On day 3, OKT3/CD28-stimulated Tcells (2.5×10⁵ cells/well) were transduced on RetroNectin® (Clontech,Mountainview, Calif.)-coated plates in the presence of IL7 and IL15. Onday 5 or 6, T cells were transferred into new wells and subsequentlyexpanded with IL-7 and IL15. Non-transduced (NT) T cells were activatedwith OKT3/CD28 and expanded in parallel with IL-7 and IL15. 47-CARexpression was determined 3 to 4 days post-transduction.

Co-Culture Assay

Recombinant Protein Co-Culture Assay.

Non-tissue culture 24-well plates were precoated with recombinant humanIL13Rα1, IL13Rα2, or IL4R protein, (R&D Systems, Minneapolis, Minn.) ata final concentration of 500 ng/well. Plates were washed once usingRPMI, and CAR or NT T cells were plated. After 24 hours, supernatantswere harvested and interferon γ (IFN γ) and Interleukin 2 (IL2) releasewere measured by ELISA according to the manufacturer's instructions (R&DSystems, Minneapolis, Minn.).

Cell Culture Co-Culture Assay.

CAR T cells were co-cultured with target cells at a 1:2 effector totarget (E:T) ratio in a 24-well plate. NT T cells served as controls.After 24 hours, culture supernatants were harvested, and the presence ofIFNγ and IL2 was determined by ELISA according to the manufacturer'sinstructions (R&D Systems, Minneapolis, Minn.).

Cytotoxicity Assay

Standard chromium (⁵¹Cr) release assays were performed as described inGottschalk et al., Blood 101:1905-1912 (2003), incorporated herein byreference. Briefly, 1×10⁶ target cells were labeled with 0.1 mCi(3.7MBq) ⁵¹Cr and mixed with decreasing numbers of effector cells togive effector to target ratios of 40:1, 20:1, 10:1, and 5:1. Targetcells incubated in complete medium alone or in 1% Triton X-100 were usedto determine spontaneous and maximum ⁵¹Cr release, respectively. After 4hours, supernatants were collected and radioactivity was measured in agamma counter (Cobra Quantum; PerkinElmer; Wellesley; MA). The meanpercentage of specific lysis of triplicate wells was calculatedaccording to the following formula: [test release−spontaneousrelease]/[maximal release−spontaneous release]×100.

Bioluminescence Imaging

Isofluorane anesthetized animals were imaged using the IVIS® system(IVIS, Xenogen Corp., Alameda, Calif.) 10-15 minutes after 150 mg/kgD-luciferin (Xenogen) per mouse was injected intraperitoneally. Thephotons emitted from the luciferase-expressing tumor cells werequantified using Living Image software (Caliper Life Sciences,Hopkinton, Mass.). A pseudo-color image representing light intensity(blue least intense and red most intense) was generated and superimposedover the grayscale reference image. Mice were euthanized when the tumorradiance was greater than 1×10⁹ on two occasions or when they meteuthanasia criteria (neurological deficits, weight loss, signs ofdistress) in accordance with the Center for Comparative Medicine atBaylor College of Medicine.

Statistics

The differences between groups were evaluated by Student's t test orone-way analysis of variance with post hoc comparison Tukey's test orDunnett's test. For the in vivo survival data, a Kaplan-Meier survivalanalysis was used, and statistical analysis was performed using a logrank test. P<0.05 was considered statistically significant.

For the experiments disclosed in Examples 13-16, the in vitroexperiments were performed at least in triplicate, and GraphPad Prism 5software (GraphPad software, Inc., La Jolla, Calif.) was used forstatistical analysis. Measurement data were presented as mean±standarddeviation (SD). The differences between means were tested by appropriatetests. The significance level used was P<0.05. For the mouseexperiments, changes in tumor radiance from baseline at each time pointwere calculated and compared between groups using t-test. Survival,determined from the time of tumor cell injection, was analyzed by theKaplan-Meier method and by the log-rank test.

Example 2 Characterization of Antigen and Screening of Hybridoma ClonesSecreting Anti-IL13Rα2 Antibodies

The primary goal of this study was to generate a high affinitymonoclonal antibody suitable for targeting of the IL13Rα2 expressed onthe surface of tumor cells. We therefore immunized mice and screened theresulting hybridoma clones for reactivity against the antigen,rhIL13Rα2, in its native conformation. A plate-bound ELISA utilizing ahybridoma clone against rhIL13Rα2, YY-23Z, was established for thedetection of rhIL13Rα2. The concentration of rhIL13Rα2 absorbed to theplastic at 1 μg/ml was found to be suitable for the detection ofantibody binding (FIG. 1A). Next, the rhIL13Rα2hFc was characterized forits “nativity” by utilizing a pair of commercially available antibodiesrecognizing only the native (found on the cell surface) and denatured(using Western blotting under reducing conditions) forms of IL13Rα2 andfor its binding properties to rhIL13Rα2 in ELISA with antibody clonesB-D13 and YY-23Z, respectively. Both clones B-D13 and YY-23Z were ableto recognize the rhIL13Rα2hFc in a plate-bound ELISA (FIG. 1B).Denaturation of antigen at 95° C. for 5 minutes in the presence ofβ-mercaptoethanol completely abolished the ability of the antibody cloneB-D13 to recognize antigen by ELISA, whereas the YY-23Z clone retainedthe ability to bind the denatured antigen. Thus, the rhIL13Rα2hFcabsorbed to the plastic of ELISA plates containing both native anddenatured forms of the protein. Analysis of serum from animals immunizedwith a fusion of rhIL13Rα2 and hFc revealed the presence of antibodiesagainst both rhIL13Rα2 and human Fc fragment. To select antibodiesspecific for the IL13Rα2 portion of the fusion, human IgG was includedas an additional negative control for the screening of hybridomapopulations. Of the 39 screened primary populations, only 15 populationswere specific to IL13Rα2, and four were reactive with human IgG.Finally, five clones strongly reacting with native IL13Rα2 were furtherexpanded and recloned. The two clones recognizing only denatured antigenwere selected from the separate immunization set with rhIL13Rα2hFcchimera. Supernatants from selected clones were compared for theirability to bind hrIL13Rα2 in a plate-bound ELISA (FIG. 1C) and byWestern blotting (FIG. 1D). FIG. 1C shows that clone 47 strongly bindsto the antigen in plate-bound ELISA but not by Western blotting,indicating the ability of clone 47 to recognize a native conformation ofthe antigen. Therefore, clone 47 was selected for furthercharacterization and for further experiments. Clone 47 was found to beof the IgG1 isotype, possessing a κ chain.

Example 3 Specificity of Binding for the IL13Rα2 (Clone 47) mAb toRecombinant Human IL13Rα2 and IL13Rα2 Expressed at the Cell Surface

We investigated the binding properties of the IL13Rα2 (clone 47) mAb torhIL13Rα2 versus the commercially available clones 83807 and B-D13 in aplate-bound ELISA. FIG. 2A shows strong and specific binding of clone 47to rhIL13Rα2 when compared with clones 83807 and B-D13. Clone 47 reachedthe plateau of binding at the low concentration of 0.05 μg/ml. None ofthe antibodies showed binding to human IgG utilized as an additionalnegative control in these experiments. To further verify the specificityof interaction for clone 47 with human IL13Rα2, a clonal line of CHOcells expressing the full size wild-type human IL13Rα2 (clone 6) wasgenerated. Binding of the antibody to control CHO cells transfected withan empty vector was compared with that of CHO cells expressing IL13Rα2.Again, the IL13Rα2 (clone 47) mAb demonstrated strong and specificbinding to IL13Rα2 expressed on the cell surface but not to control CHOcells, indicating that this antibody specifically recognizes a nativeconformation of the IL13Rα2 (FIG. 2B). Clone 47 demonstrated thestrongest affinity for IL13Rα2 at the lowest tested concentration of0.25 μg/ml. Notably, other selected hybridoma clones demonstratedsimilar specificity of interaction with IL13Rα2 expressed on the cellsurface of CHO cells but not with control CHO cells. Data obtained in aplate-bound ELISA also revealed that clone 47 does not interact with thelow affinity receptor for IL-13, the IL13Rα1 (FIG. 2C), or mouserecombinant IL13Rα2, further validating the specificity of interactionbetween clone 47 and IL13Rα2 (FIG. 2D). Clones 83807 and B-D13 did notshow binding to mouse rIL13Rα2 in agreement with current understandingof the cross-reactivity of these antibodies with mouse IL13Rα2.

We next characterized the binding capacity of clone 47 with variousglioma cell lines, the patient-derived glioma lines GBM12 and GBM43, andnormal human astrocytes. Increased expression of the IL13Rα2 generelative to normal brain tissue is reported in 44-47% of human GBMresected specimens (3) and in up to 82% (14 of 17) primary cell culturesderived from GBM and normal brain explants (2). FIGS. 3 , A and B, showthe flow charts of the comparative staining of glioma cells, humanastrocytes, and HEK cells expressing recombinant human IL13Rα2 on thecell surface with the IL13Rα2 (clones 47, 83807, and B-D13) mAb. FIGS. 3, A and B, reveal (i) various levels of IL13Rα2 expression on the cellsurface and (ii) superior binding of the clone 47 versus clones B-D13(1.2-4.6-fold difference between the cell lines) and 83807 to thesurface of analyzed cell lines. Interestingly, we observed a nearcomplete absence of the binding of clone 83807 to glioma cell lines incontrast to HEK cells expressing IL13Rα2. No binding of clone 47 wasdetected with normal human astrocytes, confirming the specificity ofinteraction of clone 47 with human glioma cells expressing IL13Rα2. Theexpression of IL13Rα2 mRNA in these cells generally correlates with thelevel of IL13Rα2 expression on the cell surface. Moreover, cellsexpressing low to no mRNA expression for IL13Rα2, including U118 andprimary human astrocytes, demonstrated low to no expression for IL13Rα2on the cell surface (FIG. 3B). In additional experiments, N10 gliomacells were incubated with either the IL13Rα2 (clone 47) mAb at 1 μg/mlor the IL13Rα2 (clone 47) mAb preincubated with a 10-fold excess ofrhIL13Rα2 (FIG. 10 ) and analyzed by flow cytometry. A significantablation of interaction between the IL13Rα2 (clone 47) mAb in thepresence of a 10-fold excess of rhIL13Rα2 was found when compared withclone 47 alone. Similarly, preincubation of N10 cells with either a10-fold excess of rhIL-13 or IL13Rα2 (clone 47) mAb almost completelyblocked the interaction between the antibody or rhIL-13 and N10 cells(supplemental FIG. 1B), indicating a specificity of recognition betweenIL13Rα2 expressed on the surface of glioma cells and clone 47 (FIG. 10).

To verify that the IL13Rα2 (clone 47) mAb possessed the ability to bindIL13Rα2 on the surface of glioma cells in situ, intracranial gliomaxenografts of U251 cells expressing green fluorescent protein (GFP) wereestablished in nude mice. Three weeks later, animals were sacrificed,and cells were obtained and placed into in vitro culture conditions.After 48 hours, the cells were collected and stained with control mIgGor IL13Rα2 (clone 47) mAb. Cultured GFP-expressing U251 cells served asa positive control. GFP-positive U251 cells represented about 56% of thetotal cells (FIG. 3C, panel a), and 96% of the cells were reactive withthe IL13Rα2 (clone 47) mAb (FIG. 3C, panel c), whereas GFP-negativecells did not interact with the antibody (FIG. 3C, panel b). These datafurther confirm that the IL13Rα2 (clone 47) mAb specifically recognizesglioma cells expressing IL13Rα2 in mouse xenografts and is not reactivewith other cells from the mouse brain.

Example 4

Affinity Studies

Surface plasmon resonance was used to determine the affinity and rate ofinteraction between the IL13Rα2 (clone 47) mAb and rhIL13Rα2. Allmeasurements were done in comparison with two commercial antibodiesagainst IL13Rα2, clones 83807 and B-D13. FIG. 4 shows the sensorgramsfor each antibody. The measurements are summarized in Table 1.

TABLE 1 Kinetics of monoclonal antibodies binding to the humanrecombinant IL13Rα2 mAbs to k_(a) k_(d) K_(D) R_(max) IL13Rα2 1/MS 1/S MRU Clone 47 9.06e4 ± 322 1.26e−4 ± 1.07e−6 1.39 × 10⁻⁹ 390 Clone 838072.23e4 ± 620 2.31e−3 ± 1.03e−5  104 × 10⁻⁹ 250 Clone B-D13   1.08e5 ±5.71e3 4.99e−3 ± 1.45e−4 46.1 × 10⁻⁹ 8-16

The estimation of kinetic parameters was performed as described inExample 1. The dissociation constant (KD) of the complexes wasdetermined as the ratio k_(a)/k_(d). For quantitative analysis, threeindependent replicates were performed for each sample. Data areexpressed as mean±S.E. These data demonstrate that the affinity ofIL13Rα2 (clone 47) mAb to recombinant IL13Rα2 exceeds the affinity ofcommercially available mAb clones 83807 and B-D13 by 75-fold and33-fold, respectively.

FIG. 4A shows that clone 47 demonstrates a prolonged and stableassociation with rhIL13Rα2 measured over a 30-minute time frame, whereasclones 83807 (FIG. 4B) and B-D13 (FIG. 4C) dissociate relativelyquickly. The affinity of binding for the IL13Rα2 (clone 47) mAb torhIL13Rα2 was calculated at 1.39×10⁻⁹M. This value exceeded the affinityof the commercially available antibody clones 83807 and B-D13 torhIL13Rα2 by 75-fold and 33-fold, respectively. Clone 47 demonstratedthe highest binding affinity (R_(max)) to rhIL13Rα2 at 390 RU whencompared with 250 and 8-16 RU for clones 83807 and B-D13, respectively.These data indicate that the IL13Rα2 (clone 47) mAb possesses propertiessuperior to clones 83807 and B-D13 as well as demonstrates a higheraffinity toward rhIL13Rα2.

Example 5

A Monoclonal Antibody Competes with rhIL-13 for Binding to IL13Rα2

To determine whether the IL13Rα2 (clone 47) mAb possesses inhibitoryproperties, competitive binding assays utilizing a rhIL13Rα2hFc chimeraand HEK cells transiently expressing the human IL13Rα2 were performed.The competitive binding assay was set up in a plate-bound ELISA format.The rhIL13Rα2hFc absorbed to the plate served as the target antigen. Todetermine whether the IL13Rα2 mAb specifically inhibits the binding ofIL-13 to rhIL13Rα2, plates were preincubated with a 100-fold excess ofmIgG, the IL13Rα2 (clone 47) mAb, or other IL13Rα2 mAb clones, including83807, YY-23Z, and B-D13, followed by incubation with rhIL13. FIG. 5Ashows that the IL13Rα2 (clone 47) mAb significantly abolished thebinding of rhIL-13 to rhIL13Rα2, whereas the IL13Rα2 mAb clones B-D13and 83807 exhibited significantly less competition for binding of humanIL-13.

To further verify the inhibitory properties of the IL13Rα2 (clone 47)mAb, HEK 293T cells were transfected with an agent encoding wild-type ora 4-amino-acid mutant form of IL13Rα2 cDNA in which Tyr207, Asp271,Tyr315, and Asp318 residues were substituted with Ala. Previously, theseresidues of the human IL13Rα2 were identified as amino acids requiredfor the interaction with the cognate ligand, IL-13. The presence of allfour mutations in one molecule has been shown to result in near completeloss of the binding of IL-13 to the mutated form of IL13Rα2 (28). After48 hours, the cells were pretreated with a 20-fold excess of rhIL-13 orthe IL13Rα2 (clone 47) mAb, followed by incubation of the IL13Rα2 (clone47) mAb or rhIL-13, respectively. FIG. 5B shows about 50% bindinginhibition of IL13Rα2 (clone 47) mAb by a 20-fold excess of rhIL-13 towild-type (WT) IL13Rα2 but not to the 4-amino-acid mutant form ofIL13Rα2. A 20-fold excess of antibody abolished the binding of rhIL-13to IL13Rα2 when expressed on the cell surface by 80%, which is similarto the result observed in plate ELISA. The residual binding of IL-13 tothe 4-amino-acid mutant form of IL13Rα2 was further decreased by anexcess of the IL13Rα2 (clone 47) mAb (FIG. 5C). Collectively, these dataindicate that the IL13Rα2 (clone 47) mAb specifically competes withrhIL-13 for the binding site on IL13Rα2. Also, these data indicate thatthe IL13Rα2 (clone 47) mAb and IL-13 have a significant overlap in theirrecognition site of the IL13Rα2 molecule.

Example 6

Role of the Tyr²⁰⁷, Asp²⁷¹, Tyr³¹⁵, and Asp³¹⁸ Residues for IL13Rα2(Clone 47) mAb Binding

Taking into consideration that IL-13 and the IL13Rα2 (clone 47)monoclonal antibody can significantly compete with one other for bindingof IL13Rα2, we determined whether the residues Tyr207, Asp271, Tyr315,and Asp318 contributing to the interaction of IL-13 with IL13Rα2 (28)were also important for binding of the IL13Rα2 (clone 47) mAb toIL13Rα2. The plasmids encoding cDNA for IL13Rα2 carrying individualmutations of Tyr207, Asp271, Tyr315, or Asp318 residues to Ala or acombination of all four mutations in one molecule were generated andtransiently expressed in HEK cells. Binding of the IL13Rα2 (clone 47)mAb to wild-type and mutant forms of IL13Rα2 was analyzed by flowcytometry. The IL13Rα2 mAbs 83807 and B-D13 were used as referenceantibodies to exclude a possible influence of variations in the level ofexpression of wild-type or mutated variants of IL13Rα2 on the surface ofHEK cells (FIG. 6A). Data were calculated as a ratio of IL13Rα2 (clone47) binding to IL13Rα2 when compared with both antibody clones 83807 andB-D13. FIG. 6A demonstrates that the binding of IL13Rα2 (clone 47) mAbwas not significantly affected by either the individual mutations or the4-amino-acid mutant form of IL13Rα2 when compared with wild-typereceptor. In contrast, binding of IL-13 to the 4-amino-acid mutant formof IL13Rα2 was nearly abolished (FIG. 6B). These data indicate that theTyr207, Asp271, Tyr315, and Asp318 residues are not crucial for theinteraction of IL13Rα2 (clone 47) mAb with IL13Rα2 but are necessary forbinding to IL-13.

Example 7

N-Linked Glycosylation Affects the Affinity of the IL13Rα2 mAb forIL13Rα2

N-Linked glycosylation has previously been demonstrated to be importantfor efficient binding of IL-13 to the cognate receptor, IL13Rα2 (30).Taking into consideration the significant overlap in epitope recognitionbetween the IL13Rα2 (clone 47) mAb and IL-13, we expected N-linkedglycosylation of IL13Rα2 to contribute to binding of the IL13Rα2 (clone47) mAb. To confirm this expectation, rhIL13Rα2hFc was treated withPngase F to remove N-linked glycosylation from the protein. The bindingof the IL13Rα2 (clone 47) mAb to control and deglycosylated targetprotein was investigated. Treatment of rhIL13Rα2 with Pngase F wasperformed under native conditions (in the absence of SDS) to avoiddenaturation of the rhIL13Rα2 affecting the binding of antibodies.Additional mAbs to IL13Rα2 (clones 83807, B-D13, and YY23Z) and rhIL-13were included in the assay to demonstrate the specificity of binding. Ina plate-bound ELISA, binding of the IL13Rα2 (clone 47) mAb to PngaseF-treated IL13Rα2 was decreased by 35% when compared with untreatedprotein (n=4; p<0.001). The binding of the IL13Rα2 (clone 83807) wasreduced by 80% when compared with untreated protein and completelyabsent for the IL13Rα2 mAbs B-D13 and YY-23Z, respectively (n=4;p<0.001) (FIG. 7A). Binding of rhIL-13 with Pngase F-treated rhIL13Rα2was also significantly diminished. To verify that Pngase F treatmentresulted in deglycosylation of the protein, control and Pngase F-treatedrhIL13Rα2hFc protein was resolved by Western blot. FIG. 7B shows thatPngase F-treated protein has a lower molecular weight, confirming theremoval of N-linked glycans from the IL13Rα2 molecule. Binding of theIL13Rα2 (clone 47) mAb to Pngase F-treated U251 glioma and HEK 293 cellsexpressing wild-type IL13Rα2 was also decreased by about 30% (n=3;p<0.05) when compared with control untreated cells (FIG. 7C).

Example 8

Immunohistochemistry

The ability of the IL13Rα2 (clone 47) mAb to detect IL13Rα2 wasevaluated in fresh frozen tissues. Flash-frozen human GBM samples or theU251 glioma flank xenograft was stained with either isotype controlmIgG1 or the IL13Rα2 (clone 47) mAb. FIG. 8 shows positive (brown)staining in the two human GBM samples, albeit with different frequencyof positive cells in the sample as well as a U251 glioma cell-basedglioma xenograft. Positive staining was detected in two of the three GBMsamples analyzed, which is consistent with the expectation that fewerthan 50% of primary GBM express IL13Rα2 (3). These data are alsoconsistent with the ability of this antibody to recognize the nativeform of IL13Rα2 expressed on the cell surface and in ELISA applications,as well as the compromised ability of this mAb to detect denaturedantigen by Western blotting.

Example 9

The IL13Rα2 Monoclonal Antibody Prolongs the Survival of Animals with anIntracranial Glioma Xenograft

The potential therapeutic properties of the IL13Rα2 (clone 47) mAb werealso determined in an orthotopic mouse model of human glioma. U251glioma cells were intracranially injected into the brain of nude micealone, in the presence of control mIgG, or with the IL13Rα2 (clone 47)mAb. FIG. 9A shows that animals in the control PBS (n=15) and mIgG(n=16) groups demonstrated a similar median survival of 27 and 25 days,respectively. In contrast, the survival of animals co-injected with theIL13Rα2 (clone 47) mAb (n=13) was significantly increased to a median of34 days (p=0.0001; mIgG versus the IL13Rα2 mAb group). Analysis of H&Estaining of the glioma xenografts from brains collected on day 17revealed a similar pattern of glioma cell distribution in the brain ofcontrol groups. In contrast, the tumor mass in the group of animalsco-injected with IL13Rα2 mAb was significantly decreased in size (FIG.9B). Independently, U251 cells were inoculated in the brains of mice and3 days later injected through the same burr hole with either PBS or theIL13Rα2 (clone 47 or B-D13) mAb as described previously (29).Interestingly, the mice injected with clone 47 demonstrated improvementin median survival when compared with PBS and clone B-D13 groups (35days versus 27 and 23 days, respectively; n=7; p>0.05) (FIG. 11 ),similar to what was found in the co-injection experiment (FIG. 9A).Nevertheless, all animals ultimately succumbed to the disease. Thesedata indicate that the IL13Rα2 (clone 47) mAb shows promise in promotingtumor rejection of IL13Rα2-expressing U251 glioma cells in the mousebrain. This finding leads to the expectation that antibody agentincorporating the IL13Rα2-binding domain of the IL13Rα2 (clone 47) mAbwill be efficacious in treating a variety of human and non-human cancerscharacterized by the presentation of IL13Rα2, such as IL13Rα2-expressingglioma cells and other malignant cell types.

References—for Examples 1-9 and for Citations Throughout the ApplicationUnless Specifically Identified Otherwise

-   1. Debinski, W., Obiri, N. I., Powers, S. K., Pastan, I., and    Puri, R. K. (1995) Clin. Cancer Res. 1, 1253-1258.-   2. Joshi, B. H., Plautz, G. E., and Puri, R. K. (2000) Cancer Res.    60, 1168-1172.-   3. Jarboe, J. S., Johnson, K. R., Choi, Y., Lonser, R. R., and    Park, J. K. (2007) Cancer Res. 67, 7983-7986.-   4. Kawakami, K., Terabe, M., Kawakami, M., Berzofsky, J. A., and    Puri, R. K. (2006) Cancer Res. 66, 4434-4442.-   5. Fujisawa, T., Joshi, B., Nakajima, A., and Puri, R. K. (2009)    Cancer Res. 69, 8678-8685.-   6. Debinski, W., Slagle, B., Gibo, D. M., Powers, S. K., and    Gillespie, G. Y. (2000) J. Neurooncol. 48, 103-111.-   7. Aman, M. J., Tayebi, N., Obiri, N. I., Puri, R. K., Modi, W. S.,    and Leonard, W. J. (1996) J. Biol. Chem. 271, 29265-29270.-   8. Gauchat, J. F., Schlagenhauf, E., Feng, N. P., Moser, R., Yamage,    M., Jeannin, P., Alouani, S., Elson, G., Notarangelo, L. D., Wells,    T., Eugster, H. P., and Bonnefoy, J. Y. (1997) Eur. J. Immunol. 27,    971-978.-   9. Akaiwa, M., Yu, B., Umeshita-Suyama, R., Terada, N., Suto, H.,    Koga, T., Arima, K., Matsushita, S., Saito, H., Ogawa, H., Fume, M.,    Hamasaki, N., Ohshima, K., and Izuhara, K. (2001) Cytokine 13,    75-84.-   10. Rahaman, S. O., Sharma, P., Harbor, P. C., Aman, M. J.,    Vogelbaum, M. A., and Haque, S. J. (2002) Cancer Res. 62, 1103-1109.-   11. Fichtner-Feigl, S., Strober, W., Kawakami, K., Puri, R. K., and    Kitani, A. (2006) Nat. Med. 12, 99-106.-   12. Shimamura, T., Fujisawa, T., Husain, S. R., Joshi, B., and    Puri, R. K. (2010) Clin. Cancer Res. 16, 577-586.-   13. Fujisawa, T., Joshi, B. H., and Puri, R. K. (2012) Int. J.    Cancer 131, 344-356.-   14. Murphy, E. V., Zhang, Y., Zhu, W., and Biggs, J. (1995) Gene    159, 131-135.-   15. Rich, T., Chen, P., Furman, F., Huynh, N., and    Israel, M. A. (1996) Gene 180, 125-130.-   16. Stupp, R., Mason, W. P., van den Bent, M. J., Weller, M.,    Fisher, B., Taphoorn, M. J., Belanger, K., Brandes, A. A., Marosi,    C., Bogdahn, U., Curschmann, J., Janzer, R. C., Ludwin, S. K.,    Gorlia, T., Allgeier, A., Lacombe, D., Cairncross, J. G.,    Eisenhauer, E., and Mirimanoff, R. O. (2005) N. Engl. J. Med. 352,    987-996.-   17. Kunwar, S., Prados, M. D., Chang, S. M., Berger, M. S., Lang, F.    F., Piepmeier, J. M., Sampson, J. H., Ram, Z., Gutin, P. H.,    Gibbons, R. D., Aldape, K. D., Croteau, D. J., Sherman, J. W., and    Puri, R. K. (2007) J. Clin. Oncol. 25, 837-844.-   18. Wykosky, J., Gibo, D. M., Stanton, C., and Debinski, W. (2008)    Clin. Cancer Res. 14, 199-208.-   19. Debinski, W., Gibo, D. M., Obiri, N. I., Kealiher, A., and    Puri, R. K. (1998) Nat. Biotechnol. 16, 449-453.-   20. Kawakami, M., Kawakami, K., and Puri, R. K. (2002) Mol. Cancer    Ther. 1, 999-1007.-   21. Husain, S. R., and Puri, R. K. (2000) Blood 95, 3506-3513.-   22. Bartolazzi, A., Nocks, A., Aruffo, A., Spring, F., and    Stamenkovic, I. (1996) J. Cell Biol. 132, 1199-1208.-   23. Kioi, M., Seetharam, S., and Puri, R. K. (2008) Mol. Cancer    Ther. 7, 1579-1587.-   24. Pini, A., and Bracci, L. (2000) Curr. Protein Pept. Sci. 1,    155-169.-   25. Ross, J. S., Gray, K., Gray, G. S., Worland, P. J., and    Rolfe, M. (2003) Am. J. Clin. Pathol. 119, 472-485.-   26. Souriau, C., and Hudson, P. J. (2003) Expert Opin. Biol. Ther.    3, 305-318.-   27. Köhler, G., and Milstein, C. (1975) Nature 256, 495-497.-   28. Arima, K., Sato, K., Tanaka, G., Kanaji, S., Terada, T., Honjo,    E., Kuroki, R., Matsuo, Y., and Izuhara, K. (2005) J. Biol. Chem.    280, 24915-24922.-   29. Sampson, J. H., Crotty, L. E., Lee, S., Archer, G. E.,    Ashley, D. M., Wikstrand, C. J., Hale, L. P., Small, C., Dranoff,    G., Friedman, A. H., Friedman, H. S., and Bigner, D. D. (2000) Proc.    Natl. Acad. Sci. U.S.A. 97, 7503-7508.-   30. Kioi, M., Seetharam, S., and Puri, R. K. (2006) FASEB J. 20,    2378-2380.-   31. McKenzie, A. N., Culpepper, J. A., de Waal Malefyt, R., Brière,    F., Punnonen, J., Aversa, G., Sato, A., Dang, W., Cocks, B. G., and    Menon, S. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 3735-3739.-   32. Donaldson, D. D., Whitters, M. J., Fitz, L. J., Neben, T. Y.,    Finnerty, H., Henderson, S. L., O'Hara, R. M., Jr., Beier, D. R.,    Turner, K. J., Wood, C. R., and Collins, M. (1998) J. Immunol. 161,    2317-2324.-   33. Hansen, H. J., Goldenberg, D. M., Newman, E. S., Grebenau, R.,    and Sharkey, R. M. (1993) Cancer 71, 3478-3485.-   34. Kuan, C. T., Wikstrand, C. J., Archer, G., Beers, R., Pastan,    I., Zalutsky, M. R., and Bigner, D. D. (2000) Int. J. Cancer 88,    962-969.-   35. Imperiali, B., and Rickert, K. W. (1995) Proc. Natl. Acad. Sci.    U.S.A. 92, 97-101.-   36. Sondermann, P., Huber, R., Oosthuizen, V., and Jacob, U. (2000)    Nature 406, 267-273.-   37. Krapp, S., Mimura, Y., Jefferis, R., Huber, R., and    Sondermann, P. (2003) J. Mol. Biol. 325, 979-989.-   38. Fernandes, H., Cohen, S., and Bishayee, S. (2001) J. Biol. Chem.    276, 5375-5383.-   39. Hsu, Y. F., Ajona, D., Corrales, L., Lopez-Picazo, J. M.,    Gurpide, A., Montuenga, L. M., and Pio, R. (2010) Mol. Cancer 9,    139.-   40. Cartron, G., Watier, H., Golay, J., and Solal-Celigny, P. (2004)    Blood 104, 2635-2642.-   41. de Haij, S., Jansen, J. H., Boross, P., Beurskens, F. J.,    Bakema, J. E., Bos, D. L., Martens, A., Verbeek, J. S., Parren, P.    W., van de Winkel, J. G., and Leusen, J. H. (2010) Cancer Res. 70,    3209-3217.-   42. Minn, A. J., Kang, Y., Serganova, I., Gupta, G. P., Giri, D. D.,    Doubrovin, M., Ponomarev, V., Gerald, W. L., Blasberg, R., and    Massagué, J. (2005) J. Clin. Investig. 115, 44-55.-   43. Kawakami, M., Kawakami, K., Kasperbauer, J. L., Hinkley, L. L.,    Tsukuda, M., Strome, S. E., and Puri, R. K. (2003) Clin. Cancer Res.    9, 6381-6388.-   44. Husain, S. R., Obiri, N. I., Gill, P., Zheng, T., Pastan, I.,    Debinski, W., and Puri, R. K. (1997) Clin. Cancer Res. 3, 151-156.-   45. Puri, R. K., Leland, P., Obiri, N. I., Husain, S. R.,    Kreitman, R. J., Haas, G. P., Pastan, I., and Debinski, W. (1996)    Blood 87, 4333-4339.-   46. Joshi, B. H., Leland, P., and Puri, R. K. (2003) Croat. Med. J.    44, 455-462.-   47. Barderas R, Bartolomé R A, Fernandez-Aceñero M J, Torres S,    Casal J I. Cancer Res. 2012 Jun. 1; 72(11):2780-90.

Example 10

A Single-Chain Antibody for Selective Targeting of IL13Rα2-ExpressingBrain Tumors

IL13Rα2 is overexpressed in a majority of high-grade astrocytomas andother malignancies, and has been validated as a target for therapeuticapplications in various preclinical models. However, current IL13-basedtherapeutic agents lack specificity due to interaction with the IL13Rα1receptor, which is widely expressed by normal or healthy cells. Thegeneration of a targeting agent that strictly binds to IL13Rα2 wouldsignificantly expand the therapeutic potential for the treatment ofIL13Rα2-expressing cancers. Recently, a monoclonal antibody 47 (mAb47)has been developed and extensively characterized. The mAb47 exclusivelybinds to a native form of human IL13Rα2. Using mAb47, a single-chainantibody (scFv) fragment was engineered from mAb47 expressed by theparental hybridoma cell line. The single-chain antibody (scFv) fragmentwas tested for its targeting properties as a soluble agent, and anadenovirus (Ad) with a modified fiber incorporating scFv47 as atargeting motif was agented.

The phage-display approach was utilized for selection of a functionalcombination of variable heavy (VH) and light (VL) chains fromestablished hybridoma cells producing mAb47. Purified phages displayingscFv47 were tested for their interaction with IL13Rα2hFc recombinantprotein, i.e., a fusion of IL13Rα2 and the Fc region of an antibody. Acompetitive ELISA was utilized to verify that the parental mAb47 and thescFv47 fragment bind to the same epitope. The soluble form of scFv47expressed in E. coli and CHO cells was analyzed by SDS-PAGE, and testedfor stability and targeting properties. To generate IL13Rα2-specific Ad,the fiber of a replication-deficient Ad5 encoding green fluorescentprotein was replaced with a chimeric fiber gene composed of a T4fibritin trimerization domain linked at its C-terminal to scFV47(AdFFscFv47-CMV-GFP). To generate viral particles, an agent encoding theadenoviral genome was rescued in HEK293F28 cells, propagated, andpurified. IL13Rα2⁺ and IL13Rα2⁻ U251 cell lines were established viastable transfection with either control or IL13Rα2-specific shRNAs(U251-IL13Rα2.KO), respectively. The AdFFscFv47-CMV-GFP virus was testedfor targeting properties in these U251 cell lines and inIL13Rα2-expressing U87 cells.

The biopanning-selected pool of phages, as well several individualclones, demonstrated specific binding to IL13Rα2hFc protein, but not tohIgG in plate ELISA. Binding of scFv47-displayed phages to IL13Rα2 wascompletely abolished by mAb47, but not by control IgG or other testedIL13Rα2 mAbs, thus confirming the same IL13Rα2 epitope was recognized byscFv47 as was recognized by the parental mAb47. Similarly tophage-displayed scFv47, the soluble scFv47 showed specific binding toIL123Rα2, but not to IL13Rα1. Interaction of Ad5FFscFv47-CMV-GFP wasalso specific to IL13Rα2-expressing U251 cells, as judged by flowcytometry for GFP expression in U251-IL13Rα2⁺ versus U251-IL13Rα2.KOcells. Furthermore, GFP expression in cells infected withAd5FFscFv47-CMV-GFP strongly correlated with the level of surfaceexpression of IL13Rα2. The specificity of viral infection was furthervalidated in a U251 glioma model.

The data validate scFv47 as a highly selective IL13Rα2 targeting agentthat provides a soluble, single-chain biologic useful in diagnosing andtreating IL13Rα2-expressing cancers, such as gliomas, colon cancers (seeExample 12) and others.

Example 11

Generation of an IL13Rα2-CAR

To generate an IL13Rα2-specific T cell, an IL13Rα2-specific chimericantigen receptor (CAR) was initially constructed. A codon-optimizedminigene was synthesized that contained the immunoglobulin heavy-chainleader peptide and the heavy and light chains of the IL13Rα2-specificsingle-chain variable fragment (scFv) separated by a linker (the scFvwas derived from hybridoma 47, Balyasnikova et al. J Biol. Chem. 2012;287(36):30215-30277). The minigene was subcloned into an SFG retroviralvector containing the human IgG1-CH2CH3 domain, a CD28 transmembranedomain, and costimulatory domains derived from CD28 and the CD3ζ-chain.CD3/CD28-activated human T cells were transduced with RD114-pseudotypedretroviral particles and subsequently expanded using IL2. Functionalanalysis revealed that T cells expressing IL13Rα2-specific CARs(IL13Rα2-CAR T cells) recognized recombinant IL13Rα2 protein as judgedby cytokine production (IFNγ and IL2; FIGS. 19 and 20 ), and killedIL13Rα2-positive cells in a cytotoxicity assay (FIG. 18 ).Non-transduced (NT) T cells did not produce cytokines and had nocytolytic activity.

Example 12

Redirecting T Cells to IL13Rα2 Positive Pediatric Glioma

IL13Rα2 is aberrantly expressed in Glioblastoma Multiforme and is,therefore, a promising target for CAR T-cell immunotherapy. The antigenrecognition domain of CARs normally consists of a single-chain variablefragment (scFv), but current IL13Rα2-specific CARs use IL13 muteins asan antigen recognition domain. IL13 mutein-based CARs, however, havebeen shown to also recognize IL13Rα1, raising significant safetyconcerns. To overcome this obstacle, a high affinity IL13Rα2-specificscFv has been agented. This scFv is used in developing a scFv-basedIL13Rα2-specific CAR (IL13Rα2-CAR), which, when expressed in T cells,will provide IL13Rα2-CAR T cells having cytotoxic effector function.

Antigen-specific T cells were incorporated into an effectiveimmunotherapy for diffuse intrinsic pontine glioma (DIPG) andglioblastoma (GBM), which are the most aggressive, uniformly fatal,primary human brain tumors in children. IL13Rα2 is expressed at a highfrequency in both DIPG and GBM, but not in normal brain, making it apromising target for T-cell immunotherapy, including scFv-based therapy,scFv-CAR T-cell-based therapy, and scFv fusions to other frameworksproviding effector function, such as BiTEs and scFv-CAR-NKs.IL13-binding CARs have been generated using mutated forms of IL13 as CARbinding domains, but these CARs also recognize IL13Rα1, raisingsignificant toxicity concerns.

To overcome this limitation, a high-affinity IL13Rα2-specific scFv thatdoes not recognize IL13Rα1 was generated. A panel of IL13Rα2-CARs wereagented that contain the IL13Rα2-specific scFv as an ectodomain, a shorthinge (SH) or a long hinge (LH), a CD28 transmembrane domain, andendodomains that contain signaling domains derived from CD3ζ andco-stimulatory molecules (e.g., CD28.ζ, CD137.ζ CD28.CD137.ζ,CD28.CD134.ζ). IL13Rα2-CAR T cells were generated by retroviraltransduction, and effector function was determined in vitro, usingco-culture and cytotoxicity assays, and in vivo, using the U373 brainxenograft model (FIG. 21 ).

Expression of all CARs in T cells was similar, as judged by Western blotanalyses. CAR cell-surface expression varied, however, depending on thehinge and endodomain of the agent. In cytotoxicity assays, the variousIL13Rα2-CAR T cells only killed target cells that expressed IL13Rα2 andnot IL13Rα1, confirming specificity (FIG. 18 ). While all IL13Rα2-CAR Tcells secreted significant levels of IFNγ in co-culture assays with theIL13Rα2⁺ glioma cell line U373 (FIG. 19 ), only short-hinge CAR T cellssecreted significant amounts of IL2 (FIG. 20 ). T cells expressingIL13Rα2-CARs with a deleted endodomain (IL13Rα2Δ-CAR) secreted nocytokines, confirming that cytokine production depends on the presenceof a functional IL13Rα2-CAR. In vivo, injection of IL13Rα2.SH.CD28ζ-CART cells into U373-bearing mice resulted in regression of gliomaxenografts, as judged by bioluminescence imaging (FIG. 21 ).IL13Rα2.LH.CD28.ζ- or IL13Rα2.Δ-CAR T cells had no antitumor effects.The data establish that a CAR that only recognizes IL13Rα2 and notIL13Rα1 was generated, and that CAR preferentially targets tumor cellsexpressing IL13Rα2. Comparison of several IL13Rα2-CARs revealed that aCAR with a SH and a CD28.ζ endodomain resulted in significant T cellactivation, as judged by IL2 production and in vivo anti-gliomaactivity. The results show that adoptive immunotherapy of primary humanbrain tumors, e.g., high-grade gliomas, in children is both feasible andpromising.

Example 13

Generation of 47-CAR T Cells

Two retroviral vectors encoding CARs based on scFv47 (47-CARs; FIG.31A)^(24,25) were initially generated. Both CARs contained an N-terminalleader sequence, a codon-optimized synthetic gene encoding scFv47, aspacer region, a CD28 transmembrane domain, and signaling domainsderived from CD28 and CD3.ζ (FIG. 31A). The spacer region was either theIgG1 hinge (16 amino acids; short spacer region (SSR);47-CAR.SSR.CD28.ζ) or the IgG1-CH2CH3 domain (293 amino acids; longspacer region (LSR); 47-CAR.LSR.CD28ζ). As controls, LSR and SSR 47-CARswithout signaling domains were constructed (47-CAR.SSR.Δ, 47-CAR.LSR.Δ;FIG. 31A). CD3/CD28-activated T cells from healthy donors weretransduced with RD114-pseudotyped retroviral particles, and 4 to 5 dayspost-transduction, T-cell phenotype and CAR expression was determined byFACS analysis. CARs were expressed on the cell surface, and thetransduction efficiency ranged from 69.2%-98.5% with no significantdifferences between constructs (FIG. 31B, C). Expression of full-length47-CAR.SSR.CD28.ζ and 47-CAR.LSR.CD28.ζ was confirmed by Western blotusing a CD3.ζ antibody for detection (FIG. 31D). Phenotypic analysisrevealed a mixture of CD4- and CD8-positive T cells. While the ratio ofCD8- to CD4-positive T cells was about 3:1 for 47-CAR.SSR.CD28.ζ,47-CAR.SSR.Δ, and 47-CAR.LSR.Δ T-cell lines, it was about 1.5:1 for47-CAR.LSR.CD28.ζ (FIG. 2 ).

Example 14

47-CAR T Cells Only Recognize IL13Rα2

To initially determine the specificity of 47-CARs, T cells expressing47-CAR.SSR.CD28.ζ, 47-CAR.LSR.CD28.ζ, M47-CAR.SSR.Δ, or M47-CAR.LSR.Δwere cultured on tissue culture plates that were uncoated or coated withrecombinant proteins encoding IL13Rα1, IL13Rα2, or IL4R. Non-transduced(NT) T cells and T cells expressing an IL13mutein-CAR.LSR.CD28.ζ10 thatrecognizes IL13Rα1 and IL13Rα2, served as controls. T cells expressing47-CAR.SSR.CD28.ζ or 47-CAR.LSR.CD28.ζ produced significant levels ofIFNγ (p<0.001) when stimulated with recombinant IL13Rα2 proteins incomparison to IL13Rα1- or IL4R-stimulated T cells (FIG. 33A). Incontrast, T cells expressing 47-CAR.SSR.Δ or 47-CAR.LSR.Δ produced noIFNγ in response to all three proteins, indicating that IFNγ productiondepends on an intact 47-CAR signaling domain. 47-CAR.LSR.CD28.ζ T cellsalso produced low levels of IFNγ without activation, indicating baselineT-cell activation, which was confirmed by intracellular staining forphosphorylated CD3.ζ (FIG. 34 ). IL13mutein-CAR.LSR.CD28.ζ T cellsproduced significant levels of IFNγ in the presence of IL13Rα1 (p<0.001)and IL13Rα2 (p<0.05) in comparison to NT T cells.

The specificity of 47-CAR T cells was then confirmed using cell linesthat were negative for IL13Rα1 and IL13Rα2 (Raji), positive for IL13Rα1(293T-GFP cells), or positive for IL13Rα1 and IL13Rα2 (U373,293T-GFP/IL13Rα2; FIG. 35 ). T cells expressing 47-CAR.SSR.CD28.ζ,47-CAR.LSR.CD28.ζ, 47-CAR.SSR.Δ, or 47-CAR.LSR.Δ were co-cultured withRaji, 293T-GFP, or 293T-GFP/IL13Rα2 cells. NT T cells served ascontrols. After 24 hours, media was collected and the concentrations ofIFNγ and IL2 were determined by ELISA. 47-CAR.SSR.CD28.ζ and47-CAR.LSR.CD28.ζ T cells produced significant amounts of IFNγ only inthe presence of U373 or 293T-GFP/IL13Rα2 cells (FIG. 33B) with SSR.CAR Tcells producing significantly more IFNγ than LSR.CAR T cells (p<0.001).47-CAR.SSR.CD28.ζ T cells produced also significant amounts of IL2 inthe presence of 293T-GFP/IL13Rα2 and U373 cells, while 47-CAR.LSR.CD28.ζT cells did not (FIG. 33C). NT-T cells and T cells expressing47-CAR.SSR.Δ or 47-CAR.LSR.Δ produced no IFNγ or IL2 in response to anytarget cells. Finally, we confirmed the specificity of 47-CAR T cells instandard cytotoxicity assays using Raji, 293T-GFP, 293T-GFP/IL13Rα2, andU373 as targets (FIG. 33D).

Example 15

Generation of Short Spacer Region (SSR) 47-CARs with CD28.OX40/41BB

While the results described above demonstrated that 47-CAR T cells onlyrecognize IL13Rα2, as judged by cytokine production and cytolyticactivity, the results also highlighted differences between LSR and SSR47-CARs. Because only 47-CAR.SSRs produced IL2 in the presence ofIL13Rα2-positive target cells, the focus in the next set of experimentswas shifted to 47-CARs with SSRs, and additional CARs were generatedwith CD28.OX40.ζ, CD28.41BB.ζ or 41BB.ζ endodomains (FIG. 36A). CAR Tcells were generated by retroviral transduction and CAR expression wasdetermined by FACS analysis (FIG. 36B, C) and Western blot (FIG. 36D).While all CARs were expressed, as judged by Western blot analysis,47-CAR.SSR.CD28.41BB.ζ was not expressed on the cell surface, and wasexcluded from further analysis.

Example 16

Comparison of Short Spacer Region 47-CARs

To compare the ability of 47-CAR.SSR T cells to produce IFNγ and IL2 inresponse to antigen exposure, co-culture assays were performed with U373cells. T cells expressing 47-CAR.SSR.Δ served as controls. All47-CAR.SSRs with functional endodomains induced IFNγ and IL2 productionin the presence of U373 cells; however, 47-CAR.SSR.41BB.ζ T cellsproduced significantly less (p<0.05) IFNγ in comparison to47-CAR.SSR.CD28.ζ and 47-CAR.SSR.CD28.OX40.ζ T cells (FIG. 37A).47-CAR.SSR.CD28.ζ T cells produced the highest amount of IL2, followedby 47-CAR.SSR.41BB.ζ and 47-CAR.SSR.CD28.OX40ζ T cells. In cytotoxicityassays, no significant difference was observed between all threeconstructs using Raji, 293T-GFP, 293T-GFP/IL13Rα2, and U373 cells astargets (FIG. 37B).

Because all three 47-CAR.SSRs T cells with functional endodomainsproduced IL2, all three constructs were tested in an orthotopic U373glioma xenograft mouse model in which T cells are directly injected intotumors.⁶ The model allows for serial bioluminescence imaging becauseU373 cells are genetically modified to express an eGFP.ffLuc fusionprotein (U373.eGFP.ffLuc). On day 0, U373.eGFP.ffLuc cells were injectedstereotactically into brains of SCID mice and, on day 7, T cellsexpressing 47-CAR.SSR.CD28.ζ, 47-CAR.SSR.41BB.ζ, 47-CAR.SSR.CD28.OX40.ζor 47-CAR.SSR.Δ were injected intratumorally. While mice treated with47-CAR.SSR.Δ T cells showed continuous tumor growth within 4 days ofT-cell injection, mice treated with 47-CAR.SSR T cells that hadfunctional endodomains did not (FIG. 38A, B). Comparison ofbioluminescence imaging results revealed no significant differencebetween 47-CAR.SSR.Δ T cells and the 47-CAR.SSR T cells groups on theday of T-cell injection. Mice treated with 47-CAR.SSR.CD28.ζ or47-CAR.SSR.CD28.OX40.ζ T cells, however, had significantly lower tumorsignals as early as one day post-treatment in comparison to mice treatedwith 47-CAR.SSR.Δ T cells (p=0.012; Table 2). This resulted in asignificant survival advantage for 47-CAR.SSR.CD28.ζ or47-CAR.SSR.CD28.OX40.ζ T-cell-treated mice (p=0.0002 and p=0.0092; FIG.40C). While 47-CAR.SSR.41BB.ζ T-cell-treated mice responded slower,resulting in a significant difference between 47-CAR.SSR.Δ T-celltreated on day 14 (p=0.005; Table 2), treatment with this CAR T cellalso resulted in a significant survival advantage (p=0.0039; FIG. 5CFIG. 40C). 47-CAR.SSR.CD28.ζ T-cell-treated mice had the longest mediansurvival (84 days). There was no statistical difference, however, incomparison to the median survival of 47-CAR.SSR.41BB.ζ (63 days) or47-CAR.SSR.CD28.OX404 (56 days) T-cell-treated mice.

TABLE 2 Tumor signal comparison P^(†) Day 7 SSR.Δ vs. SSR.41BB.ζ 0.917SSR.Δ vs. SSR.CD28.ζ 0.111 SSR.Δ vs. SSR.CD28.OX40.ζ 0.917 Day 8 SSR.Δvs. SSR.41BB.ζ 0.835 SSR.Δ vs. SSR.CD28.ζ 0.012 SSR.Δ vs.SSR.CD28.OX40.ζ 0.023 Day 14 SSR.Δ vs. SSR.41BB.ζ 0.005 SSR.Δ vs.SSR.CD28.ζ 0.015 SSR.Δ vs. SSR.CD28.OX40.ζ 0.015 Day 21 SSR.Δ vs.SSR.41BB.ζ 0.010 SSR.Δ vs. SSR.CD28.ζ 0.010 SSR.Δ vs. SSR.CD28.OX40.ζ0.012 Day 28 SSR.Δ vs. SSR.41BB.ζ 0.051 SSR.Δ vs. SSR.CD28.ζ 0.008 SSR.Δvs. SSR.CD28.OX40.ζ 0.034 ^(†)Wilcoxon rank-sum test

While 47-CAR T cells had potent anti-glioma activity, mice developedrecurrent gliomas. To investingate the etiology of tumor recurrence,U373 cells were isolated from two tumor-bearing mice that had beentreated either with 47-CAR.SSR.CD28.ζ or 47-CAR.SSR.CD28.OX40ζ T cells.FACS analysis after short-term culture revealed cell surface expressionof IL13Rα2, and these cells were readily killed by 47-CART cells incytotoxicity assays (FIG. 39 ). Next, the persistence of T-cells wasdetermined by genetically modifying T cells with 47-CAR.SSR.CD28.ζ andeGFP.ffLuc (Luc/47-CAR T cells), and injecting them into U373tumor-bearing mice. T cells persisted for less than 7 days. Withoutwishing to be bound by theory, limited persistence appears to be themost likely explanation for tumor recurrence (FIG. 40 ).

References for Examples 13-16

-   1. Okada, H, Kohanbash, G, Zhu, X, Kastenhuber, E R, Hoji, A, Ueda,    R et al. (2009). Immunotherapeutic approaches for glioma. Crit Rev    Immunol 29: 1-42.-   2. Suryadevara, C M, Verla, T, Sanchez-Perez, L, Reap, E A, Choi, B    D, Fecci, P E et al. (2015). Immunotherapy for malignant glioma.    Surg Neurol Int 6: S68-S77.-   3. Van Gool, S W (2015). Brain Tumor Immunotherapy: What have We    Learned so Far? Front Oncol 5: 98.-   4. Reardon, D A, Freeman, G, Wu, C, Chiocca, E A, Wucherpfennig, K    W, Wen, P Y et al. (2014). Immunotherapy advances for glioblastoma.    Neuro Oncol 16: 1441-1458.-   5. Ahmed, N, Salsman, V S, Kew, Y, Shaffer, D, Powell, S, Zhang, Y J    et al. (2010). HER2-specific T cells target primary glioblastoma    stem cells and induce regression of autologous experimental tumors.    Clin Cancer Res 16: 474-485.-   6. Chow, K K, Naik, S, Kakarla, S, Brawley, V S, Shaffer, D R, Yi, Z    et al. (2013). T Cells Redirected to EphA2 for the Immunotherapy of    Glioblastoma. Mol Ther 21: 629-637.-   7. Johnson, L A, Scholler, J, Ohkuri, T, Kosaka, A, Patel, P R,    McGettigan, S E et al. (2015). Rational development and    characterization of humanized anti-EGFR variant III chimeric antigen    receptor T cells for glioblastoma. Sci Transl Med 7: 275ra22.-   8. Brown, C E, Badie, B, Barish, M E, Weng, L, Ostberg, J R, Chang,    W C et al. (2015). Bioactivity and Safety of IL13Ralpha2-Redirected    Chimeric Antigen Receptor CD8+ T cells in Patients with Recurrent    Glioblastoma. Clin Cancer Res-   9. Brown, C E, Starr, R, Aguilar, B, Shami, A F, Martinez, C,    D'Apuzzo, M et al. (2012). Stem-like tumor-initiating cells isolated    from IL13Ralpha2 expressing gliomas are targeted and killed by    IL13-zetakine-redirected T Cells. Clin Cancer Res 18: 2199-2209.-   10. Krebs, S, Chow, K K, Yi, Z, Rodriguez-Cruz, T, Hegde, M, Gerken,    C et al. (2014). T cells redirected to interleukin-13Ralpha2 with    interleukin-13 mutein-chimeric antigen receptors have anti-glioma    activity but also recognize interleukin-13Ralpha1. Cytotherapy 16:    1121-1131.-   11. Kong, S, Sengupta, S, Tyler, B, Bais, A J, Ma, Q, Doucette, S et    al. (2012). Suppression of human glioma xenografts with    second-generation IL13R-specific chimeric antigen receptor-modified    T cells. Clin Cancer Res 18: 5949-5960.-   12. Choi, B D, Suryadevara, C M, Gedeon, P C, Herndon Ii, J E,    Sanchez-Perez, L, Bigner, D D et al. (2013). Intracerebral delivery    of a third generation EGFRvIII-specific chimeric antigen receptor is    efficacious against human glioma. J Clin Neurosci.-   13. Kawakami, M, Kawakami, K, Takahashi, S, Abe, M, Puri, R K    (2004). Analysis of interleukin-13 receptor alpha2 expression in    human pediatric brain tumors. Cancer 101: 1036-1042.-   14. Okada, H, Low, K L, Kohanbash, G, McDonald, H A, Hamilton, R L,    Pollack, I F (2008). Expression of glioma-associated antigens in    pediatric brain stem and non-brain stem gliomas. J Neurooncol 88:    245-250.-   15. Joshi, B H, Puri, R A, Leland, P, Varricchio, F, Gupta, G,    Kocak, M et al. (2008). Identification of interleukin-13 receptor    alpha2 chain overexpression in situ in high-grade diffusely    infiltrative pediatric brainstem glioma. Neuro Oncol 10: 265-274.-   16. Brown, C E, Warden, C D, Starr, R, Deng, X, Badie, B, Yuan, Y C    et al. (2013). Glioma IL13Ralpha2 is associated with mesenchymal    signature gene expression and poor patient prognosis. PLoS One 8:    e77769.-   17. Hsi, L C, Kundu, S, Palomo, J, Xu, B, Ficco, R, Vogelbaum, M A    et al. (2011). Silencing IL-13Ralpha2 promotes glioblastoma cell    death via endogenous signaling. Mol Cancer Ther 10: 1149-1160.-   18. Fichtner-Feigl, S, Strober, W, Kawakami, K, Puri, R K, Kitani, A    (2006). IL-13 signaling through the IL-13alpha2 receptor is involved    in induction of TGF-beta1 production and fibrosis. Nat Med 12:    99-106.-   19. Dotti, G, Gottschalk, S, Savoldo, B, Brenner, M K (2014). Design    and development of therapies using chimeric antigen    receptor-expressing T cells. Immunol Rev 257: 107-126.-   20. Sadelain, M, Brentjens, R, Riviere, I (2013). The basic    principles of chimeric antigen receptor design. Cancer Discov 3:    388-398.-   21. Jensen, M C, Riddell, S R (2015). Designing chimeric antigen    receptors to effectively and safely target tumors. Curr Opin Immunol    33: 9-15.-   22. Brown, C E, Starr, R, Aguilar, B, Shami, A, Martinez, C,    D'Apuzzo, M et al. (2012). Stem-like tumor initiating cells isolated    from IL13Ralpha2-expressing gliomas are targeted and killed by    IL13-zetakine redirected T cells. Clin Cancer Res-   23. Kahlon, K S, Brown, C, Cooper, L J, Raubitschek, A, Forman, S J,    Jensen, M C (2004). Specific recognition and killing of glioblastoma    multiforme by interleukin 13-zetakine redirected cytolytic T cells.    Cancer Res 64: 9160-9166.-   24. Balyasnikova, I V, Wainwright, D A, Solomaha, E, Lee, G, Han, Y,    Thaci, B et al. (2012). Characterization and immunotherapeutic    implications for a novel antibody targeting interleukin (IL)-13    receptor alpha2. J Biol Chem 287: 30215-30227.-   25. Kim, J, Young, J, Solomaha, E, Kanojia, D, Lesniak, M S,    Balyasnikova, I V (2015). A novel single-chain antibody redirects    adenovirus to IL13Rα2-expressing brain tumors. (submitted for    publication)-   26. Brown, C E, Starr, R, Naranjo, A, Wright, C, Bading, J, Ressler,    J et al. (2011). Adoptive Transfer of Autologous    IL13-zetakine+Engineered T Cell Clones for the Treatment of    Recurrent Glioblastoma: Lessons from the Clinic. Molecular Therapy    19: S136-S137.-   27. Beard, R E, Abate-Daga, D, Rosati, S F, Zheng, Z, Wunderlich, J    R, Rosenberg, S A et al. (2013). Gene expression profiling using    nanostring digital RNA counting to identify potential target    antigens for melanoma immunotherapy. Clin Cancer Res 19: 4941-4950.-   28. Kunwar, S, Prados, M D, Chang, S M, Berger, M S, Lang, F F,    Piepmeier, J M et al. (2007). Direct intracerebral delivery of    cintredekin besudotox (IL13-PE38QQR) in recurrent malignant glioma:    a report by the Cintredekin Besudotox Intraparenchymal Study Group.    J Clin Oncol 25: 837-844.-   29. Okada, H, Kalinski, P, Ueda, R, Hoji, A, Kohanbash, G, Donegan,    T E et al. (2011). Induction of CD8+ T-cell responses against novel    glioma-associated antigen peptides and clinical activity by    vaccinations with {alpha}-type 1 polarized dendritic cells and    polyinosinic-polycytidylic acid stabilized by lysine and    carboxymethylcellulose in patients with recurrent malignant glioma.    J Clin Oncol 29: 330-336.-   30. Iwami, K, Shimato, S, Ohno, M, Okada, H, Nakahara, N, Sato, Y et    al. (2012). Peptide-pulsed dendritic cell vaccination targeting    interleukin-13 receptor alpha2 chain in recurrent malignant glioma    patients with HLA-A*24/A*02 allele. Cytotherapy-   31. Kioi, M, Seetharam, S, Puri, R K (2008). Targeting    IL-13Ralpha2-positive cancer with a novel recombinant immunotoxin    composed of a single-chain antibody and mutated Pseudomonas    exotoxin. Mol Cancer Ther 7: 1579-1587.-   32. Haso, W, Lee, D W, Shah, N N, Stetler-Stevenson, M, Yuan, C M,    Pastan, I H et al. (2013). Anti-CD22-chimeric antigen receptors    targeting B-cell precursor acute lymphoblastic leukemia. Blood 121:    1165-1174.-   33. Hudecek, M, Lupo-Stanghellini, M T, Kosasih, P L, Sommermeyer,    D, Jensen, M C, Rader, C et al. (2013). Receptor affinity and    extracellular domain modifications affect tumor recognition by    ROR1-specific chimeric antigen receptor T cells. Clin Cancer Res 19:    3153-3164.-   34. Guest, R D, Hawkins, R E, Kirillova, N, Cheadle, E J, Arnold, J,    O'Neill, A et al. (2005). The role of extracellular spacer regions    in the optimal design of chimeric immune receptors: evaluation of    four different scFvs and antigens. J Immunother 28: 203-211.-   35. Kunkele, A, Johnson, A J, Rolczynski, L S, Chang, C A, Hoglund,    V, Kelly-Spratt, K S et al. (2015). Functional Tuning of CARs    Reveals Signaling Threshold above Which CD8+ CTL Antitumor Potency    Is Attenuated due to Cell Fas-FasL-Dependent AICD. Cancer Immunol    Res 3: 368-379.-   36. Hoyos, V, Savoldo, B, Quintarelli, C, Mahendravada, A, Zhang, M,    Vera, J et al. (2010). Engineering CD19-specific T lymphocytes with    interleukin-15 and a suicide gene to enhance their    anti-lymphoma/leukemia effects and safety. Leukemia 24: 1160-1170.-   37. Wels, W, Harwerth, I M, Zwickl, M, Hardman, N, Groner, B, Hynes,    N E (1992). Construction, bacterial expression and characterization    of a bifunctional single-chain antibody-phosphatase fusion protein    targeted to the human erbB-2 receptor. Biotechnology (N Y) 10:    1128-1132.-   38. Pule, M A, Straathof, K C, Dotti, G, Heslop, H E, Rooney, C M,    Brenner, M K (2005). A chimeric T cell antigen receptor that    augments cytokine release and supports clonal expansion of primary    human T cells. Mol Ther 12: 933-941.-   39. Vera, J, Savoldo, B, Vigouroux, S, Biagi, E, Pule, M, Rossig, C    et al. (2006). T lymphocytes redirected against the kappa light    chain of human immunoglobulin efficiently kill mature B    lymphocyte-derived malignant cells. Blood 108: 3890-3897.-   40. Xu, Y, Zhang, M, Ramos, C A, Durett, A, Liu, E, Dakhova, O et    al. (2014). Closely related T-memory stem cells correlate with in    vivo expansion of CAR.CD19-T cells and are preserved by IL-7 and    IL15. Blood 123: 3750-3759.-   41. Gottschalk, S, Edwards, O L, Sili, U, Huls, M H, Goltsova, T,    Davis, A R et al. (2003). Generating CTL against the subdominant    Epstein-Barr virus LMP1 antigen for the adoptive Immunotherapy of    EBV-associated malignancies. Blood 101: 1905-1912.

Example 17

Transgenic Expression of IL15 Increases 47-CAR T-Cell PersistenceResulting in Enhanced Anti-Tumor Activity

IL13Rα2-CAR.CD28.ζ T cells expressing IL15 (IL13Rα2-CAR.IL15 T cells)were generated by double transducing T cells with retrovirusescontaining expression cassettes encoding i) IL13Rα2-CAR.CD28.ζ or ii)IL15, A Nerve Growth Factor Receptor (ΔNGFR), and inducible Caspase 9(iC9) separated by 2A sequences. Suitable 2A sequences include any 2Asequence known in the art, as exemplified by the 2A amino acid sequencefrom porcine teschovirus-1 (SEQ ID NO:109) encoded by the polynucleotidesequence set forth as SEQ ID NO:110, the 2A amino acid sequence fromThoseaasigna virus (SEQ ID NO:111) encoded by the polynucleotidesequence set forth as SEQ ID NO:112, the 2A amino acid sequence fromEquine rhinitis A virus (ERAV) (SEQ ID NO:113) encoded by thepolynucleotide sequence set forth as SEQ ID NO:114, or the 2A amino acidsequence from Foot and Mouth Disease Virus (FMDV) (SEQ ID NO:115)encoded by the polynucleotide sequence set forth as SEQ ID NO:116. Kimet al., PLoS One 6(4):1-8 (2011). The effector function ofIL13Rα2-CAR.IL15 T cells was determined in vitro using standard assays,and in the U373 GBM xenograft model.

Double transduction of CD3/CD28-activated T cells resulted in T-celllines that expressed both transgenes in 45-50% of T cells. At base line,IL13Rα2-CAR.IL15 T cells produced on average 69.5 pg/ml of IL15.Production was significantly increased after CD3 or antigen-specificT-cell stimulation (176.7 pg/ml; n=6; p<0.001). IL13Rα2-CAR.IL15 T cellswere as efficient as IL13Rα2-CAR T cells in killing IL13Rα2-positive GBMcells in vitro. After intratumoral injection into U373 glioma-bearingmice, IL13Rα2-CAR.IL15 T cells persisted significantly longer thanIL13Rα2-CAR T cells (p<0.05). This resulted in a significant increase inprogression-free (98 versus 49 days; p=0.004) and overall survival(p=0.006) of treated mice.

The data disclosed in this Example demonstrate that transgenicexpression of IL15 enhances the in vivo persistence of IL13Rα2-CAR Tcells, resulting in improved anti-glioma activity.

Each of the references cited herein is hereby incorporated by referencein its entirety or in relevant part, as would be apparent from thecontext of the citation.

From the disclosure herein it will be appreciated that, althoughspecific embodiments of the disclosure have been described herein forpurposes of illustration, various modifications may be made withoutdeviating from the spirit and scope of the disclosure.

What is claimed is:
 1. A IL13Rα2-specific CAR comprising the amino acidsequence of SEQ ID NO: 53 or SEQ ID NO:
 55. 2. A pharmaceuticalcomposition comprising the IL13Rα2-specific CAR of claim
 1. 3. A methodof treating a cancer that expresses IL13Rα2 in a subject, comprisingadministering to the subject a population of cells comprising theIL13Rα2-specific CAR of claim 1, in an amount effective to treat thecancer in the subject.
 4. The method of claim 3, wherein the populationof cells are T-lymphocytes or natural killer cells.
 5. The method ofclaim 3, wherein the cancer comprises glioblastoma.